CN107418559A - A kind of bacteriophage electrochemical luminescence signals amplification probe and its preparation method and application - Google Patents
A kind of bacteriophage electrochemical luminescence signals amplification probe and its preparation method and application Download PDFInfo
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- CN107418559A CN107418559A CN201710598361.4A CN201710598361A CN107418559A CN 107418559 A CN107418559 A CN 107418559A CN 201710598361 A CN201710598361 A CN 201710598361A CN 107418559 A CN107418559 A CN 107418559A
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Abstract
The invention discloses a kind of bacteriophage electrochemical luminescence signals amplification probe and its preparation method and application.Described bacteriophage electrochemical luminescence signals amplification probe includes M13 bacteriophages, amalgamation and expression the Streptavidin in M13 bacteriophages, and is covalently attached the tris (bipyridine) ruthenium on M13 bacteriophages.Bacteriophage electrochemical luminescence signals amplification probe of the present invention, the connecting framework amplified using M13 bacteriophages as tris (bipyridine) ruthenium signal, with stable system, it is not susceptible to assemble, and the advantages that easily preserving, obtained amplification of signal probe has the advantages that simple, controllable, signal amplification efficiency is high, the quantitative detection available for nucleic acid or antigen etc..
Description
Technical field
The invention belongs to biochemical analysis technical field, specifically, is related to a kind of bacteriophage electrochemical luminescence signals amplification
Probe and its preparation method and application.
Background technology
Accurately, sensitive, specific bioanalysis functional molecular (such as nucleic acid, protein) is in life science and biomedicine
The clinical diagnosis field of research and disease tool is of great significance.Since 21 century, multidisciplinary synthesis, nucleic acid are had benefited from
Probe and sensing technology have obtained deeper, broader development.
Existing nucleic acid detection technique depends on nucleic acid amplification technologies, such as PCR (PCR), nucleic acid
The amplification (NASBA) of sequence dependent, ring mediated isothermal amplification (LAMP), rolling circle amplification (RCA) etc., by the way that these amplifications are produced
The detection techniques such as thing combination fluorescence, chemiluminescence or electrochemical luminescence, it is possible to achieve highly sensitive nucleic acid detects.However, this
The problem of a little methods are present is that they are not direct detection methods, but by carrying out exponential amplification to nucleic acid to carry out
Detection.Therefore, the mistake or the height of efficiency occurred in amplification process, directly affects the reading of testing result
Take, it is difficult to quantitative detection is carried out to sample.
Based on this, invent a kind of directly nucleic acid detection method and it is quantified, there is important scientific value.It is existing
In some methods, mainly by the strategy being amplified to the signal of detection probe, such as by the way that signal probe is tagged to
In nano material (nanogold, graphene, CNT etc.), based on multiple marker sites in nano material, realize that signal is put
Greatly.But these methods are there is also some problems, including：1) the bio-compatible sex chromosome mosaicism of nano material, the biology of nano material
Stability is generally poor, is easily focused in some biosystems (such as hypersaline environment), and influences the reappearance of detection；2)
The mark of nano material is typically uncontrollable because the synthesis to nano material at present be difficult to controllable precise (including its
Size, pattern etc.), this directly affects nano material and the controllability of bioprobe mark, so that the detection essence of probe
Degree is difficult to the requirement for reaching clinical detection.
The content of the invention
The shortcomings that in order to overcome prior art and deficiency, primary and foremost purpose of the invention are to provide a kind of bacteriophage electrochemistry hair
Optical signal amplification probe, it has the advantages that simple, controllable, signal amplification efficiency is high.
It is a further object of the present invention to provide the preparation method of the bacteriophage electrochemical luminescence signals amplification probe.
Another object of the present invention is to provide the bacteriophage electrochemical luminescence signals amplification probe in bioanalysis
Using.
The purpose of the present invention is achieved through the following technical solutions：
A kind of bacteriophage electrochemical luminescence signals amplification probe, it includes M13 bacteriophages, amalgamation and expression in M13 bacteriophages
In Streptavidin, and be covalently attached tris (bipyridine) ruthenium on M13 bacteriophages.
Bacteriophage electrochemical luminescence signals amplification probe of the present invention, believed using M13 bacteriophages as tris (bipyridine) ruthenium
The connecting framework of number amplification, has stable system, is not susceptible to assemble, and the advantages that easily preserve, obtained amplification of signal
Probe has the advantages that simple, controllable, signal amplification efficiency is high.
M13 bacteriophages are a kind of filamental bacteriophages, nontoxic, and five p3 albumen positioned at the small capsid in one end participate in
The identification process of biological virus, and the p8 albumen for being located at the main capsid of side wall is only responsible for the morphosis of bacteriophage.Each M13 phagocytosis
Body about expresses 2000 p8 albumen, therefore can carry out abundant chemical modification on the p8 albumen of M13 bacteriophages, and this repaiies
Decorations do not interfere with the function of M13 phage virus identification antigen.Traditional inorganic nano-particle amplification of signal probe is typically only capable to
Realize 100 times or so of amplification of signal, and on the p8 albumen of M13 bacteriophages can mark more than 1000 tris (bipyridine) ruthenium point
Son, it is thus possible to more than 1000 times of amplification of signal is realized, obtains higher signal amplification efficiency, can be by electrochemical luminescence signals
The sensitivity of detection improves more than two orders of magnitude.
The preparation method of bacteriophage electrochemical luminescence signals amplification probe of the present invention, it comprises the following steps：
1) terpyridyl Ruthenium-N-hydroxy-succinamide ester (Ru (bpy) is prepared3 2+-NHS)；
2) by Streptavidin amalgamation and expression on M13 bacteriophages, Streptavidin-M13 bacteriophages are made；
3) by terpyridyl Ruthenium-N-hydroxy-succinamide ester (Ru (bpy)3 2+- NHS) be covalently attached to Streptavidin-
On M13 bacteriophages, bacteriophage electrochemical luminescence signals amplification probe (i.e. Streptavidin-M13 phagocytosis of the present invention is obtained
Body-ruthenium amplification of signal probe).
Further, above-mentioned steps 1) specifically include：With cis- dichloro two (2,2 '-bipyridyl) ruthenium (II) and 2,2 '-connection pyrrole
Pyridine -4,4 '-dicarboxylic acids are raw material, are reacted in methanol/water in the mixed solvent, obtain carboxylated tris (bipyridine) ruthenium, i.e. ruthenium-two
(2,2 '-bipyridyl) (2,2 '-bipyridyl -4,4 '-dicarboxylic acids)；Sodium hexafluoro phosphate is then added, obtains tris (bipyridine) ruthenium hexafluoro phosphorus
Hydrochlorate (Ru (bpy)2(dcbpy)(PF6)2), i.e. double (hexafluoros of ruthenium-two (2,2 '-bipyridyl) (2,2 '-bipyridyl -4,4 '-dicarboxylic acids)
Phosphate)；Then n-hydroxysuccinimide (NHS) and N, N are added '-dicyclohexylcarbodiimide (DCC), with N, N '-diformazan
Base formamide (DMF) is that solvent is reacted, and obtains terpyridyl Ruthenium-N-hydroxy-succinamide ester (Ru (bpy)3 2+-NHS)。
Further, above-mentioned steps 2) specifically include：By on the gene fragment clone of Streptavidin to phagemid vector,
Then convert to Escherichia coli and cultivated, take positive colony plasmid, add helper phage and cultivated, culture terminates laggard
Row centrifugation, obtains Streptavidin-bacteriophage.Wherein, described phagemid vector is phagemid vector pC, phagemid vector
P12 or phagemid vector p87；Described helper phage is helper phage M13KO7.
Phagemid vector pC, phagemid vector p12 and phagemid vector p87 are the phagemid vector of M13 bacteriophages, will
After the genetic fragment of Streptavidin is connected on above-mentioned phagemid vector, SA-PIII fusion proteins can be formed, in signal peptide
Under guiding, the cell membrane of M13 bacteriophages can be passed through to enter pericentral siphon chamber, the assembling and release of M13 bacteriophages is participated in, be thus connected to
On the p3 albumen of M13 bacteriophages.
Further, above-mentioned steps 2) in, by Streptavidin amalgamation and expression on the P3 albumen of M13 bacteriophages, each
1~5 Streptavidin molecule is expressed on M13 bacteriophages.Each M13 bacteriophages there are about 3~5 P3 albumen, by by strepto-
Avidin is fused on P3 albumen, and each M13 bacteriophages can show 1~5 Streptavidin molecule.
Further, above-mentioned steps 3) specifically include：Streptavidin-M13 the bacteriophages that step 2) is obtained add
Cultivate, centrifuge in PEG/NaCl mixed solutions, obtained bacteriophage bead is resuspended in sodium bicarbonate buffer liquid, then adds step
The rapid terpyridyl Ruthenium-N-hydroxy-succinamide ester 1) obtained is reacted, and after purified, obtained Streptavidin-M13 and is bitten
Thalline-ruthenium amplification of signal probe.
Further, above-mentioned steps 3) in, terpyridyl Ruthenium-N-hydroxy-succinamide ester is covalently attached to M13 and bitten
On the lysine amino of the P8 albumen of thalline.The amino of the lysine of the P8 albumen of M13 bacteriophages can be with terpyridyl Ruthenium-N-
Hydroxysuccinimide eater (Ru (bpy)3 2+- NHS) NHS groups condensation reaction occurs, so as to by Ru (bpy)3 2+Covalently
It is connected on the P8 albumen of M13 bacteriophages.Each M13 bacteriophages contain about 2000 P8 albumen, therefore each M13 phagocytosis physical efficiencys
The Ru (bpy) that enough marks is more than 10003 2+Molecule.
A kind of nucleic acid-bacteriophage electrochemical luminescence signals probe, by bacteriophage electrochemical luminescence signals of the present invention
The DNA or rna probe of amplification probe and biotin labeling are formed.
Nucleic acid of the present invention-bacteriophage electrochemical luminescence signals probe, the detection available for target nucleic acid sequence.
Nucleic acid of the present invention-bacteriophage electrochemical luminescence signals probe, by by the DNA or RNA of biotin labeling
Probe is combined with Streptavidin-bacteriophage-ruthenium amplification of signal probe of the present invention, the chain based on high specific, high-affinity
Mould Avidin-Biotin interaction principle, obtain a kind of nucleic acid-bacteriophage-ruthenium signal probe.Nucleic acid-the bacteriophage-ruthenium letter
Number probe with target nucleic acid by being hybridized, and after separating treatment, hybrid product addition electrochemiluminescence cell is detected,
Electrochemical luminescence signals value is read, carries out quantitative detection to target nucleic acid by the analysis to electrochemical luminescence signals intensity.
A kind of antibody-bacteriophage electrochemical luminescence signals probe, by bacteriophage electrochemical luminescence signals of the present invention
The antibody probe of amplification probe and biotin labeling is formed.
Antibody of the present invention-bacteriophage electrochemical luminescence signals probe, the detection available for target antigen.
Antibody of the present invention-bacteriophage electrochemical luminescence signals probe, by by the antibody probe of biotin labeling
Combined with Streptavidin-bacteriophage-ruthenium amplification of signal probe of the present invention, the strepto- parent based on high specific, high-affinity
With element-biotin interaction principle, a kind of antibody-bacteriophage-ruthenium signal probe is obtained.Antibody-bacteriophage-ruthenium the signal probe
By being specifically bound with target antigen, after separating treatment, Ag-Ab product addition electrochemiluminescence cell is examined
Survey, read electrochemical luminescence signals value, carry out quantitative detection to target antigen by the analysis to electrochemical luminescence signals intensity.
Brief description of the drawings
Fig. 1 is the synthetic route chart of Streptavidin-M13 bacteriophages-ruthenium amplification of signal probe；
Fig. 2 is the spectral characterization figure of Streptavidin-M13 bacteriophages-ruthenium amplification of signal probe；
Fig. 3 is the nucleic acid detection method schematic diagram of nucleic acid-bacteriophage electrochemical luminescence signals probe；
Fig. 4 is that (concentration of sample 1 is 0pM, sample for the detection of nucleic acids result figure of nucleic acid-bacteriophage electrochemical luminescence signals probe
The concentration of product 2 is 1pM, and the concentration of sample 3 is 5pM, and the concentration of sample 4 is 25pM, and the concentration of sample 5 is 125pM)；
Fig. 5 is that (concentration of sample 1 is 0pM, sample for the antigen testing result figure of antibody-bacteriophage electrochemical luminescence signals probe
The concentration of product 2 is 0.5pM, and the concentration of sample 3 is 2.5pM, and the concentration of sample 4 is 12.5pM, and the concentration of sample 5 is 62.5pM).
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
Embodiment one：Prepare terpyridyl Ruthenium-N-hydroxy-succinamide ester (Ru (bpy)3 2+-NHS)
1st, tris (bipyridine) ruthenium hexafluorophosphate (Ru (bpy) is synthesized2(dcbpy)(PF6)2)
By the cis- dichloros two of 0.2g (2,2 '-bipyridyl) ruthenium (II), 0.15g 2,2 '-bipyridyl -4,4 '-dicarboxylic acids, 0.2g
Sodium acid carbonate, 32mL methanol and 8mL deionized waters are added in three-neck flask, condenser are installed, under magnetic stirring, in silicone oil
It is heated to 80 DEG C in bath, and heating reflux reaction 10 hours, reaction solution is gradually changed into orange red from puce.After reaction terminates
The pH value of reaction solution is adjusted to 4.4 with the concentrated sulfuric acid, cooled 2 hours in ice bath under dark surrounds, by excess 2,2 '-
Bipyridyl -4,4 '-dicarboxylic acids Precipitation, then vacuumizing filtration, collects filtrate, obtains carboxylated tris (bipyridine) ruthenium filtrate.
The hexafluorophosphoric acid sodium solution that 12.5mL concentration is 0.2g/mL is added into carboxylated tris (bipyridine) ruthenium filtrate, after stirring 5 minutes,
Cool 2 hours in ice bath, form maroon sediment.Solution and sediment are transferred in 15mL centrifuge tubes, at 4 DEG C with
5000rmp is centrifuged 5 minutes, collects maroon sediment, tris (bipyridine) ruthenium hexafluorophosphate is obtained after freeze-drying.
2nd, terpyridyl Ruthenium-N-hydroxy-succinamide ester (Ru (bpy)3 2+-NHS)
0.12g n-hydroxysuccinimides (NHS) and 0.23g N, N '-dicyclohexylcarbodiimide (DCC) are dissolved in
2mL N, in N '-dimethyl formamide (DMF), stirring, cooled in 0 DEG C of ice-water bath, then add 0.19g terpyridyls
Ruthenium hexafluorophosphate, reaction 5 hours is stirred at room temperature after 30 minutes in stirring reaction at 0 DEG C, and all reactants are needed first through nothing
Water process.After reaction terminates, centrifuged 5 minutes with 5000rmp at 4 DEG C, collect supernatant, after freeze-drying, obtain product three
Bipyridyl Ruthenium-N-hydroxy-succinamide ester.Product is placed in Brown Glass Brown glass bottles and jars only, protected in the environment of freezing, dry, lucifuge
Deposit standby.
Embodiment two：Prepare Streptavidin-M13 bacteriophages
1st, specific primer is designed, the DNA fragmentation of Streptavidin (SA) is cloned on phagemid vector pC, obtains matter
Grain pC-SA.
Following (the SEQ ID NO in such as sequence table of the DNA sequence dna of Streptavidin (SA)：Shown in 1)：
atggacccctccaaggactcgaaggcccaggtctcggctgccgaggccggcatcaccggcacctggtacaaccagct
cggctcgaccttcatcgtgaccgcgggcgccgacggcgccctgaccggaacctacgagtcggccgtcggcaacgccg
agagccgctacgtcctgaccggtcgttacgacagcgccccggccaccgacggcagcggcaccgccctcggttggacg
gtggcctggaagaataactaccgcaacgcccactccgcgaccacgtggagcggccagtacgtcggcggcgccgaggc
gaggatcaacacccagtggctgctgacctccggcaccaccgaggccaacgcctggaagtccacgctggtcggccacg
acaccttcaccaaggtgaagccgtccgccgcctccatcgacgcggcgaagaaggccggcgtcaacaacggcaacccg
ctcgacgccgttcagcag。
Sense primer：tataatGGCCCAGCCGGCC(underscore part is SfiI enzymes to ATGGCCATGGACCCCTCCAAG
Enzyme site)；
Anti-sense primer：ataaaTGCGGCCGCCTGCTGAACGGCGTC (underscore part is NotI restriction enzyme sites).
Using the DNA sequence dna of Streptavidin as template, in the presence of above-mentioned specific primer, protected using KOD Plus are high
True property polymerase kit (spinning (Shanghai) bio tech ltd purchased from Japan), is carried out to the DNA fragmentation of Streptavidin
PCR is expanded；Then SfiI and NotI restriction enzymes are used, respectively the DNA fragmentation to Streptavidin and phagemid vector pC
After carrying out digestion, it is attached with DNA ligase, obtains plasmid pC-SA.
PCR reaction systems are following (50 μ l)：
Pcr amplification reaction condition is：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 68 DEG C of extension 30s,
Reaction carries out 30 circulations altogether；Last 68 DEG C of extensions 5min.
Endonuclease reaction system is following (50 μ l)：
Endonuclease reaction condition：50 DEG C of water-bath 4 hours, room temperature cooling is placed in, adds 1 μ l restriction enzyme Not I,
37 DEG C of water-baths 4 hours.
Coupled reaction system is following (50 μ l)：
Coupled reaction condition：25 DEG C of incubation reactions 30 minutes.
2nd, plasmid pC-SA is converted to Escherichia coli and cultivated, obtain Streptavidin-M13 bacteriophages.
10ng plasmid pC-SA are taken, are added to the ER2738 competent cells that 100 μ L are prepared, on ice incubation 0.5 hour, so
After be placed in 42 DEG C of water-baths, be incubated 90 seconds, then be placed in and be incubated 2 minutes on ice.Add 500 μ L 2 × YT culture mediums, in 37 DEG C,
150rpm is cultivated 4 hours.50 μ L samples are taken, 200 μ L is diluted to, is coated on 2 × YT-AG flat boards, are inverted in 37 DEG C of incubator trainings
Support overnight.Picking monoclonal, being inoculated in 2 × YT-AG of 5mL culture mediums, (A is ammonia benzyl sistomycocin, final concentration of 100 μ g/mL；G is
Glucose, it is final concentration of 2%), in 37 DEG C, 210rpm overnight incubations.
Overnight bacterium is inoculated into 100mL 2 × YT-AG culture mediums, OD600 is reached 0.1；Cultivated in 37 DEG C, 210rpm
1.5~2.5 hours, OD600 is set to reach 0.4.It is subsequently placed in 2 minutes on ice, adds 30 μ L helper phage M13KO7, mixes
It is even, cultivated 0.5 hour in 30 DEG C, 50rpm.Final concentration of 50 μ g/mL kanamycins is added, in 30 DEG C, 210rpm cultures 0.5
Hour.Nutrient solution is transferred to sterile centrifuge bottles, centrifuged 15 minutes under 4 DEG C, 1600rpm；Supernatant is abandoned, addition is preheated to 37 DEG C
2 × YT-AK culture mediums (A is ammonia benzyl sistomycocin, final concentration of 100 μ g/mL；K is kanamycins, final concentration of 50 μ g/mL)
Precipitation thalline is resuspended, then in 30 DEG C, 210rpm overnight incubations.Nutrient solution is transferred to sterile centrifuge bottles, in 4 DEG C, 8000rpm
Lower centrifugation 20 minutes, collects supernatant, is filtered with 0.45 μM of filter, obtains Streptavidin-M13 bacteriophages.
Embodiment three：Prepare Streptavidin-M13 bacteriophages-ruthenium amplification of signal probe
By 100 μ L 1012Pf μ/mL Streptavidin-M13 bacteriophages, add 20 μ L PEG/NaCl mixed solutions
Mixed in (20%w/v PEG8000/2.5M NaCL), be placed in and cultivate 1 hour on ice, then centrifuged under room temperature 11000rpm
20 minutes, obtain M13 bacteriophage beads.M13 bacteriophage beads are resuspended in 100 μ L 0.2M pH8.3 NaHCO3It is slow
In fliud flushing, 5 μ L 10mg/mL terpyridyl Ruthenium-N-hydroxy-succinamide ester (Ru (bpy) is added3 2+- NHS), it is black at 4 DEG C
Oscillation incubation is stayed overnight under dark situation.10 μ L 1.5M pH8.5 hydroxylamine solution is added in M13 phage solutions, room temperature cultivates 1
Hour, so as to which end mark is reacted.Then filtered by PEG precipitations and the centrifugal desalting columns of Zeba, remove unreacted three
Pyridine Ruthenium-N-hydroxy-succinamide ester.After purified, Streptavidin-M13 bacteriophages-ruthenium amplification of signal probe is obtained, such as
Shown in Fig. 2, its spectral signature figure presents the characteristic absorption peak of tris (bipyridine) ruthenium at 450nm.
Example IV：The structure of nucleic acid-M13 bacteriophages-ruthenium signal probe and its application in genetic test
1st, nucleic acid-M13 bacteriophages-ruthenium signal probe is built
According to the pre-S gene gene orders of HBV viruses, design nucleic acid signal probe 5 '-
GCTCAGTTTACTAGTGCCATTT- biotins -3 '.By 10 μ L 100nM nucleic acid signal probe 5 '-
GCTCAGTTTACTAGTGCCATTT- biotins -3 ', 10 μ L 100nM Streptavidin-M13 bacteriophages-ruthenium amplification of signal
Probe, add in 80 μ L PBS, be well mixed, be incubated, obtain final concentration of 10nM nucleic acid-M13 bacteriophages-ruthenium
Signal probe.
2nd, magnetic bead-Gene traps probe is built
According to the pre-S gene gene orders of HBV the viruses ,-COOH- of design Gene traps probe 5 '
TTTTTGGAGCACCCACGTGTCCTGGCC-3′.The magnetic bead of carboxyl modified is taken, with 100 μ L 100mM pH7.0 imidazole buffer
Liquid washs three times, then adds 500 μ L 100mM imidazole buffer (including 30mM EDC), gently concussion 30 minutes, then
- COOH-TTTTTGGAGCAC the CCACGTGTCCTGGCC-3 ' of 500nM Gene traps probe 5 ' are added, in the case where gently shaking, in
37 DEG C are incubated 2 hours.Then washed with 100 μ L cleaning fluids (7mM Tris-HCl+pH8.0170mM NaCl+0.05%Tween20)
Wash three times, be resuspended in 500 μ L buffer solutions (20mM Tris-HCl+pH8.0500mM NaCl).
3rd, probe hybridization and electrochemical luminescence detection
Take 10 μ L nucleic acid-M13 bacteriophages-ruthenium signal probe, 10 μ L magnetic beads-Gene traps probe, 10 μ L differences dense respectively
Degree the-ACTAGTAAACTGAGCATACTGGCCAGGACACGTGGGTGC-3 ' of pre-S gene genes target sequence 5 ' (0pM,
1pM, 5pM, 25pM, 125pM), 10 μ 5 × PBSs of L are added, it is 50 μ L to add water to cumulative volume.Hybridize 30 points in 75 DEG C
Clock, then recover to 37 DEG C, hybrid product is added into electrochemiluminescence cell carries out enrichment with magnetic bead, cleaning, then adds tripropyl amine (TPA)
Buffer solution carries out electrochemical luminescence detection.The electrochemical luminescence signals value of each hybrid product, testing result such as Fig. 4 institutes are read respectively
Show.Testing result shows that the detection sensitivity of this probe reaches 1pM.
According to the electrochemical luminescence signals value of this method measure gene samples, according to the electrochemistry of the target sequence of normal concentration
Luminous signal value is calculated, you can quantitative detection is carried out to testing gene sample.
Embodiment five：The structure of antibody-M13 bacteriophages-ruthenium signal probe and its application in antigen detection
1st, antibody-M13 bacteriophages-ruthenium signal probe is built
Buy commercialized biotin-Cea Monoclonal Antibodies.By 10 μ L 100nM biotin-carcinomebryonic antigen list
Streptavidin-M13 bacteriophages-ruthenium amplification of signal probe of clonal antibody, 10 μ L 100nM, add 80 μ L PBS
In, it is well mixed, is incubated, obtains final concentration of 10nM antibody-M13 bacteriophages-ruthenium signal probe.
2nd, magnetic bead-antibody capture probe is built
Buy commercialized biotin-anti-carcinoembryonic antigen and catch antibody.By 10 μ L 500nM biotin-anti-carcinoembryonic antigen
Catch antibody, the Streptavidin coating magnetic bead that 1mg particle diameters are 2.8 μm, add in 90 μ L PBS, be well mixed, incubate
Educate, obtain final concentration of 50nM magnetic bead-antibody capture probe.
3rd, probe hybridization and electrochemical luminescence detection
Take 10 μ L antibody-M13 bacteriophages-ruthenium signal probe, 10 μ L magnetic beads-antibody capture probe, 10 μ L differences dense respectively
The carcinomebryonic antigen (0pM, 0.5pM, 2.5pM, 12.5pM, 62.5pM) of degree, 10 μ 5 × PBSs of L are added, add water to totality
Product is 50 μ L.It is incubated 20 minutes in 37 DEG C, product will be incubated and add electrochemiluminescence cell progress enrichment with magnetic bead, cleaning, Ran Houjia
Enter tripropyl amine (TPA) buffer solution and carry out electrochemical luminescence detection.The electrochemical luminescence signals value of each incubation product, detection knot are read respectively
Fruit is as shown in Figure 5.Testing result shows that the detection sensitivity of this probe reaches 0.5pM.
According to the electrochemical luminescence signals value of this method measure antigen samples, sent out according to the electrochemistry of the antigen of normal concentration
Optical signal value is calculated, you can antigen samples to be measured are carried out with quantitative detection.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Sequence table
<110>Guangzhou is rich to come this bio tech ltd
<120>A kind of bacteriophage electrochemical luminescence signals amplification probe and its preparation method and application
<160> 3
<170> PatentIn version 3.1
<210> 1
<211> 480
<212> DNA
<213>Artificial sequence
<220>
<223>Streptavidin gene
<400> 1
atggacccct ccaaggactc gaaggcccag gtctcggctg ccgaggccgg 50
catcaccggc acctggtaca accagctcgg ctcgaccttc atcgtgaccg 100
cgggcgccga cggcgccctg accggaacct acgagtcggc cgtcggcaac 150
gccgagagcc gctacgtcct gaccggtcgt tacgacagcg ccccggccac 200
cgacggcagc ggcaccgccc tcggttggac ggtggcctgg aagaataact 250
accgcaacgc ccactccgcg accacgtgga gcggccagta cgtcggcggc 300
gccgaggcga ggatcaacac ccagtggctg ctgacctccg gcaccaccga 350
ggccaacgcc tggaagtcca cgctggtcgg ccacgacacc ttcaccaagg 400
tgaagccgtc cgccgcctcc atcgacgcgg cgaagaaggc cggcgtcaac 450
aacggcaacc cgctcgacgc cgttcagcag 480
<210> 2
<211> 40
<212> DNA
<213>Artificial sequence
<220>
<223>Streptavidin upstream region of gene primer
<400> 2
tataatggcc cagccggcca tggccatgga cccctccaag 40
<210> 3
<211> 29
<212> DNA
<213>Artificial sequence
<220>
<223>Streptavidin downstream of gene primer
<400> 3
ataaatgcgg ccgcctgctg aacggcgtc 29
Claims (9)
- A kind of 1. bacteriophage electrochemical luminescence signals amplification probe, it is characterised in that：Including M13 bacteriophages, amalgamation and expression in M13 Streptavidin in bacteriophage, and it is covalently attached the tris (bipyridine) ruthenium on M13 bacteriophages.
- 2. the preparation method of the bacteriophage electrochemical luminescence signals amplification probe described in claim 1, comprises the following steps：1) terpyridyl Ruthenium-N-hydroxy-succinamide ester is prepared；2) by Streptavidin amalgamation and expression on M13 bacteriophages, Streptavidin-M13 bacteriophages are made；3) terpyridyl Ruthenium-N-hydroxy-succinamide ester is covalently attached on Streptavidin-M13 bacteriophages, obtains this The described bacteriophage electrochemical luminescence signals amplification probe of invention.
- 3. preparation method according to claim 2, it is characterised in that the step 1) specifically includes：With cis- dichloro two (2,2 '-bipyridyl) ruthenium (II) and 2,2 '-bipyridyl -4,4 '-dicarboxylic acids are raw material, are carried out in methanol/water in the mixed solvent anti- Should, obtain carboxylated tris (bipyridine) ruthenium；Sodium hexafluoro phosphate is then added, obtains tris (bipyridine) ruthenium hexafluorophosphate；Then add N-hydroxysuccinimide and N, N '-dicyclohexylcarbodiimide, with N, N '-dimethyl formamide is that solvent is reacted, and is obtained To terpyridyl Ruthenium-N-hydroxy-succinamide ester.
- 4. preparation method according to claim 2, it is characterised in that the step 2) specifically includes：By Streptavidin Gene fragment clone on phagemid vector, then convert to Escherichia coli and cultivated, take positive colony plasmid, add auxiliary Helper phage is cultivated, and culture is centrifuged after terminating, and obtains Streptavidin-bacteriophage；The phagemid vector is to bite Bacterium grain carrier pC, phagemid vector p12 or phagemid vector p87；The helper phage is helper phage M13KO7.
- 5. the preparation method according to claim 2 or 4, it is characterised in that：In the step 2), Streptavidin is merged Expression expresses 1~5 Streptavidin molecule on the P3 albumen of M13 bacteriophages on each M13 bacteriophages.
- 6. preparation method according to claim 2, it is characterised in that the step 3) specifically includes：Step 2) is obtained Streptavidin-M13 bacteriophages add in PEG/NaCl mixed solutions and cultivate, centrifuge, obtained bacteriophage bead is resuspended in In sodium bicarbonate buffer liquid, then add terpyridyl Ruthenium-N-hydroxy-succinamide ester that step 1) obtains and reacted, passed through After purification, Streptavidin-M13 bacteriophages-ruthenium amplification of signal probe is obtained.
- 7. the preparation method according to claim 2 or 6, it is characterised in that：In the step 3), by terpyridyl Ruthenium-N- Hydroxysuccinimide eater is covalently attached on the lysine amino of P8 albumen of M13 bacteriophages.
- A kind of 8. nucleic acid-bacteriophage electrochemical luminescence signals probe, as the bacteriophage electrochemical luminescence signals described in claim 1 The DNA or rna probe of amplification probe and biotin labeling are formed.
- A kind of 9. antibody-bacteriophage electrochemical luminescence signals probe, as the bacteriophage electrochemical luminescence signals described in claim 1 The antibody probe of amplification probe and biotin labeling is formed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108107037A (en) * | 2017-12-20 | 2018-06-01 | 华南师范大学 | Food-borne pathogens detection method based on nuclease protection procedures and electrochemical luminescence principle |
| CN111996238A (en) * | 2020-08-12 | 2020-11-27 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | New virus trace detection method using electrochemical luminescence scanning imaging system |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060110723A1 (en) * | 2000-12-11 | 2006-05-25 | Genetrace Systems, Inc. | Multiplexed protein expression and activity assay |
| CN102731622A (en) * | 2012-06-07 | 2012-10-17 | 清华大学深圳研究生院 | Quantum dot-phage complex of specific targeting human embryonic stem cell |
| CN103113877A (en) * | 2013-01-11 | 2013-05-22 | 长春理工大学 | Fluorescent probe for detecting microcystic toxin-LR in water |
| CN106319032A (en) * | 2015-06-24 | 2017-01-11 | 深圳华大基因研究院 | Method for measuring activity of Phi29 DNA polymerase and rolling circle sequencing method |
| CN107057683A (en) * | 2016-12-28 | 2017-08-18 | 浙江大学 | A kind of preparation method of fibroin fluorescence probe |
-
2017
- 2017-07-20 CN CN201710598361.4A patent/CN107418559B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060110723A1 (en) * | 2000-12-11 | 2006-05-25 | Genetrace Systems, Inc. | Multiplexed protein expression and activity assay |
| CN102731622A (en) * | 2012-06-07 | 2012-10-17 | 清华大学深圳研究生院 | Quantum dot-phage complex of specific targeting human embryonic stem cell |
| CN103113877A (en) * | 2013-01-11 | 2013-05-22 | 长春理工大学 | Fluorescent probe for detecting microcystic toxin-LR in water |
| CN106319032A (en) * | 2015-06-24 | 2017-01-11 | 深圳华大基因研究院 | Method for measuring activity of Phi29 DNA polymerase and rolling circle sequencing method |
| CN107057683A (en) * | 2016-12-28 | 2017-08-18 | 浙江大学 | A kind of preparation method of fibroin fluorescence probe |
Non-Patent Citations (3)
| Title |
|---|
| HYO-EON JIN: "Collagen mimetic peptide engine ered M13 bacterio phage for collagen targeting and imaging in cancer", 《BIOMATERIALS》 * |
| 刘敏 等: "生物传感器探针在致病菌检测中的研究进展", 《安徽农业科学》 * |
| 曲青蓉: "重组噬菌体靶向探针在动脉血栓近红外活体成像中的应用", 《东南大学 博士学位论文》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108107037A (en) * | 2017-12-20 | 2018-06-01 | 华南师范大学 | Food-borne pathogens detection method based on nuclease protection procedures and electrochemical luminescence principle |
| CN111996238A (en) * | 2020-08-12 | 2020-11-27 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | New virus trace detection method using electrochemical luminescence scanning imaging system |
| CN111996238B (en) * | 2020-08-12 | 2024-04-05 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | New virus trace detection method using electrochemiluminescence scanning imaging system |
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