CN108467883A - Antibody detection method associated with a kind of triple strand dna molecular beacon combination nano-pore technology - Google Patents
Antibody detection method associated with a kind of triple strand dna molecular beacon combination nano-pore technology Download PDFInfo
- Publication number
- CN108467883A CN108467883A CN201810198020.2A CN201810198020A CN108467883A CN 108467883 A CN108467883 A CN 108467883A CN 201810198020 A CN201810198020 A CN 201810198020A CN 108467883 A CN108467883 A CN 108467883A
- Authority
- CN
- China
- Prior art keywords
- antibody
- strand dna
- single strand
- combined area
- molecular beacon
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
Abstract
The invention discloses antibody detection methods associated with a kind of triple strand dna molecular beacon combination nano-pore technology.Inventor has designed and synthesized the triple strand dna molecular beacon for detecting antibody first, and after antibody is combined with molecular beacon, molecular beacon is destructurized, discharges probe；Antibodies Antibodies capture DNA molecular compound can not pass through nano-pore since volume is excessive, and probe passes through nano-pore will will produce characteristic current signal, and the signal specificity is extremely strong, can accurately realize that molecule is pointed out, the problem of avoiding background interference.In addition, the unique thermodynamic and kinetic properties of molecular beacon are combined with this single molecule techniques of nano-pore, the high sensitivity detection to the antibody that appraises at the current rate is realized.Moreover, method of the invention also has easy to operate, and reagent consumption is few, the advantages that without large-scale instrument.
Description
Technical field
The present invention relates to antibody detection methods associated with a kind of triple strand dna molecular beacon combination nano-pore technology.
Background technology
For antibody as a kind of important biochemical indicator, rapid sensitive detection is significant not only for medical diagnosis on disease,
It is the important means of observation of curative effect and disease prevention.The antibody test technology generally used at present mainly has enzyme linked immunosorbent assay
With two kinds of immunoblotting.But both methods is not only complicated for operation, consumes a large amount of reagents, and obtained result is generally also
All it is semi-quantitative results.In recent years with the rapid development of nanotechnology, the various nano materials that are based on are developed in succession to realize
The method of highly sensitive, highly selective detection antibody.But there is still a need for by large-scale instrument and process it is cumbersome.Therefore develop it is simple,
Efficient antibody detection method becomes more urgent.Since DNA has base pair complementarity, can be carried out in base or chain end more
It plants the advantages such as modification and the different nano-machines of various functions can be designed to.2015, Ricci et al. utilized end modified
The molecular beacon structure of antigen and fluorophor realizes the quick and easy detection of high sensitivity of DigiTAb (divalent).With
The method for modifying fluorophor based on molecular beacon afterwards realizes the detection of Multiple Antibodies (divalent) again, makes this method widely people
Know, but still bad for the detection result for the antibody that appraises at the current rate.Further, since this method depend on optical signalling, therefore there is
The problems such as photobleaching, fluorescence lifetime is short, background interference, especially background interference easily obtains false positive conclusion.Therefore research is opened
The antibody detection method for sending out highly sensitive, highly selective, the low background interference based on molecular beacon is highly advantageous to medical industry
Development.
Nano-pore single channel technology was developed on the basis of electrophysiologic studies from middle 1990s
The emerging detection means got up.So-called nano-pore is exactly aperture size in the duct of nanoscale, usually 1-100 nanometers.When
The compartment of two mutually insulateds full of electrolyte passes through nanoscale hole link, under DC Electric Field, the electricity in solution
It solves matter ion directional migration and across nano-pore to generate electric current, measures through Patch Clamp System, is recorded after amplification and conversion.
When, there are when substance to be detected, which passes through nano-pore under diffusion or voltage driving, leads in duct at this time in solution
The ion populations crossed generate variation due to occupying for determinand, change so as to cause the electric current being recorded, current signal
Including two characteristic quantities：Electric current blocks amplitude (amplitude) and electric current residence time (dwell time), can therefrom analyze
Obtain abundant physical property infomation：Type, structure, conformation change and molecular composition of substance etc..Generally, bion nano-pore
Sensitivity and signal-to-noise ratio it is more much better than artificial material nano-pore, therefore biological nano hole is more advantageous to the highly sensitive inspection of realization
It surveys.But biological nano hole is got by plasmid expression, geometric dimension has been fixed, therefore only size is matched
Molecule can just pass through.The scale of antibody can not pass through α HL usually in ten rans.JACS is delivered within 2011
The work of cocaine is detected based on aptamer combination HL nano-pores.It is compound that Y types can be formed after aptamer combination cocaine
Structure.The structure can not pass through α HL nano-pores and long-time current blockage occurs, and this blocking current signal exactly also is utilized
Cocaine is realized quickly to detect.But jam signal is highly detrimental to highly sensitive detection, and because its signal characteristic is low, the back of the body
Scape interference is stronger.
Invention content
The object of the present invention is to provide antibody test sides associated with a kind of triple strand dna molecular beacon combination nano-pore technology
Method.
Present invention firstly provides a kind of molecular combinations for detecting target antibody, including probe molecule and antibody capture
Molecule；
The probe molecule is to modify melon ring to obtain in the 5 ' ends of single strand dna I；In single strand dna I,
3 ' ends have the combined area A being made of n nucleotide；
The antibody capture molecule is made of single strand dna II and two antigen molecules；During single strand dna II is located at
Between, two ends of single strand dna II are separately connected an antigen molecule corresponding with purpose antibody；In DNA molecular II, 5 '
Hold the combined area B for having and being made of n nucleotide, 3 ' ends that there is the combined area C being made of n nucleotide；
Combined area B and combined area C is reverse sequence；Combined area B and combined area A is reverse complementary sequence.
The present invention also protects a kind of compound for detecting target antibody, is incubated altogether by probe molecule and antibody capture molecule
It educates to be formed；
The probe molecule is to modify melon ring to obtain in the 5 ' ends of single strand dna I；In single strand dna I,
3 ' ends have the combined area A being made of n nucleotide；
The antibody capture molecule is made of single strand dna II and two antigen molecules；During single strand dna II is located at
Between, two ends of single strand dna II are separately connected an antigen molecule corresponding with purpose antibody；In DNA molecular II, 5 '
Hold the combined area B for having and being made of n nucleotide, 3 ' ends that there is the combined area C being made of n nucleotide；
Combined area B and combined area C is reverse sequence；Combined area B and combined area A is reverse complementary sequence.
Concretely 4 DEG C of the reaction condition that the probe molecule and antibody capture molecule are incubated altogether, 2h.
It is incubated altogether after the probe molecule and the mixing of antibody capture molecule equimolar.
Random natural numbers of any description above n between 6-10.
The length of any description above single strand dna I is 18-22bp.
The length of any description above single strand dna II is 33-41bp.
Any description above n is 8.
The length of any description above single strand dna I is 20bp.
The length of any description above single strand dna II is 37bp.
The compound that melon ring can be connect by any description above with DNA molecular is ferrocene.
In any description above single strand dna I, the nucleotide in addition to the A of combined area is C.
The combined area A concretely GAGAGAGA.
In any description above single strand dna II, connected by N number of identical base among the combined area B and combined area C
It connects.Random natural numbers of the N between 19-23.The N concretely 21.By N number of among the combined area B and combined area C
T connections.
The combined area B concretely TCTCTCTC.
The combined area C concretely CTCTCTCT.
Any description above single strand dna I specifically can be as shown in the sequence 1 of sequence table.
Any description above single strand dna II is specific as shown in the sequence 2 of sequence table.
In any description above probe molecule, melon ring is modified by linking arm in the 5 ' ends of single strand dna I.
The linking arm is specifically formed by phosphate group, triazole and ferrocene.
In any description above probe molecule, each element is sequentially connected below：Single strand dna I, phosphate group, three
Nitrogen azoles, ferrocene and melon ring.Melon ring is connect by Subjective and Objective supermolecular mechanism with ferrocene.
Any description above melon ring concretely cucurbit(7)uril.
The preparation method of any description above probe molecule specifically comprises the following steps：
(a1) 3.3 μ L single strand dna I solution (solvent be water, a concentration of 100 μM of single strand dna I), 1.2 μ L are taken
Deionized water, 2 μ L nitrine solution of ferrocene (solvent is acetonitrile, a concentration of 200mM of nitrine ferrocene), 1 μ L sodium ascorbates
Solution (solvent is water, a concentration of 20mM of sodium ascorbate), 0.5 μ L copper nitrate solutions (solvent is water, copper nitrate it is a concentration of
20mM), 2 μ L HEPES buffer solutions (200mM) mix, mixed solution totally 10 μ L.
(a2) mixed solution that step (a1) obtains is vortexed at room temperature and reacts 2h, 2 μ L EDTA solution are then added
(100mM) terminates reaction.
(a3) after completing step (a2), using 6 gel exclusion chromatography columns of Micro bio-spin, by specification operate into
Row desalination.
(a4) 10 μ L melons rings { Q [7] } (5mM) are added in the sample obtained after step (a3) desalination and are incubated 2h, obtain probe.
Originally a kind of system of protection, including any molecular combinations and sample cell system are returned；
The sample cell system includes two compartments, and two compartment polytetrafluoroethylene films separate；The polytetrafluoroethylene film
Centre tool is there are one a diameter of 100-150 μM of through-hole, and the through-hole is full of by phospholipid bilayer, in the phospholipid bilayer
The nano-pore formed by alpha hemolysin heptamer albumen there are one in layer；It is formed using Ag/AgCl electrodes in sample cell system
Closed circuit.
The present invention also protects a kind of system, including any compound and sample cell system；
The sample cell system includes two compartments, and two compartment polytetrafluoroethylene films separate；The polytetrafluoroethylene film
Centre tool is there are one a diameter of 100-150 μM of through-hole, and the through-hole is full of by phospholipid bilayer, in the phospholipid bilayer
The nano-pore formed by alpha hemolysin heptamer albumen there are one in layer；It is formed using Ag/AgCl electrodes in sample cell system
Closed circuit.
The thickness of the polytetrafluoroethylene film is 20 μm.
The volume of described two compartments (cis and trans) is 1.5mL.
The construction method of the sample cell system is specific as follows：
The mixed solution that 1 parts by volume hexadecane and 10 parts by volume pentanes form is instilled into polytetrafluoro with capillary glass tube
Around the aperture of vinyl film (dripping quantity is about 2 to 3 microlitres), electrolyte buffer liquid (3M then will be added in two compartments
KCl, 10mM Tris, pH 5.0) and 1, (1,2- bis- phytane acyl lecithin is with phosphatide original for bis- phytane acyl lecithin (1ipids) of 2-
The form of liquid is added, and a concentration of 10 mg/ml of phosphatide stoste, addition is about 15 microlitres；The addition of electrolyte buffer liquid
Amount is 1mL), liquid-transfering gun mixing is used in combination, it is made to be self-assembly of phospholipid bilayer.Then it is added 1 into the solution of cis compartments
Microlitre wild type alpha hemolysin heptamer protein solution (concentration of protein solution is about 0.3 mg/ml), the spontaneous insertion of albumen
Phospholipid bilayer forms nano-pore, and (high frequency sampling probe is connected with, high frequency sampling probe is connected with using Ag/AgCl electrodes
Patch clamp amplifier) in sample cell system form closed circuit.
The present invention also protects the application of any description above molecular combinations or compound or system in testing goal antibody.
The present invention also protects a kind of method detecting purpose antibody in sample, includes the following steps：By sample to be tested with
Upper any compound is added after being incubated jointly in the sample cell system of any description above, special by analysing whether to generate
Levy detection of the electric signal realization to purpose antibody.
In the method, the reaction condition of the incubation is 25 DEG C of incubation 30min.
In the method, any description above is added after sample to be tested and the compound of any description above are incubated jointly
Sample cell system in after, complex concentration in detection architecture concretely 200nM.
The signal acquisition carries out under the conditions of 25 ± 2 DEG C of room temperature.
Signal sampling frequencies：100kHz, Bessel low-pass filtering are by frequency：10kHz.
The analysis of patch-clamp current signal is analyzed using 10.2 softwares of Clampfit, characteristic signal manual extraction and hand
It is dynamic to measure, data are handled and are fitted using Origin 8.5.
If being able to detect that characteristic signal, illustrates to contain purposeful antibody in sample to be tested, can further be believed by feature
Number occur frequency the antibody in sample to be tested is quantified；If can't detect characteristic signal, illustrate in sample to be tested not
Containing purposeful antibody.
Any description above purpose antibody specific can be Dig antibody (Abeam companies, article No.：Ab6212), 5BrdU antibody
(Abcam companies, article No.：Ab8152), HIV p17 antibody (Abcam companies, article No.：Ab9067), Streptavidin (Sigma-
Aldrich, article No.：Or dig fab fragement (Roche companies, article No. 85878)：11093274910).
When purpose antibody is Dig antibody, the antigen molecule is digoxin molecule, the antibody capture molecule such as formula
(1) shown in.
When purpose antibody is 5BrdU antibody, the antigen molecule is 5BrdU bases, the antibody capture molecule such as formula
(2) shown in.
When purpose antibody is HIV p17 antibody, the antigen molecule is antigen molecule (p17 polypeptides：N-terminal-
The ends ELDRWEKIRLRP-C), in the antibody capture molecule, the 5 ' ends and 3 ' ends of single strand dna II are more with p17 respectively
Peptide connects.
When purpose antibody is Streptavidin, the antigen molecule is biotin, the antibody capture molecule such as formula (3)
It is shown.
When purpose antibody is dig fab fragement antibody, the antigen molecule is digoxin molecule, the capture
Shown in element such as formula (1).
The present inventor devises triple strand dna molecular beacon and is combined it with nano-pore technology, by dividing
The sub- end modified different antigen molecule of beacon constructs the detection method of corresponding antibody.In the embodiment of the present invention, inventor
DNA probe molecule has been synthesized first, it is incubated jointly with antibody capture DNA molecular forms triple strand dna molecular beacon later.When
After antibody is combined with molecular beacon, molecular beacon is destructurized, and DNA probe molecule is released.Antibody-antibody captures
DNA molecular compound can not pass through nano-pore since volume is excessive, and DNA probe molecule passes through nano-pore will will produce feature
Current signal, the signal specificity is extremely strong, the problem of can accurately realizing that molecule is pointed out, avoid background interference.In addition, point
The sub- unique thermodynamic and kinetic properties of beacon are combined with this single molecule techniques of nano-pore, are realized to the antibody that appraises at the current rate
High sensitivity detection.Moreover, method of the invention also has easy to operate, and reagent consumption is few, without big
The advantages that type instrument.
Description of the drawings
Fig. 1 is the principle schematic and three stranded DNA structure design that triple strand dna detects antibody.
Fig. 2 is that dig antibody concentrations detect working curve.
Fig. 3 is that BrdU antibody concentrations detect working curve.
Fig. 4 is that HIV p17 antibody concentrations detect working curve.
Fig. 5 is dig fab fragement Concentration Testing working curves.
Fig. 6 is Streptavidin Concentration Testing working curve.
Fig. 7 is dig antibody selective enumeration method experimental results.
Fig. 8 is BrdU antibody selective enumeration method experimental results.
Fig. 9 is HIV p17 antibody selective enumeration method experimental results.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Probe obtains the modification of melon ring in the 5 ' ends of single strand dna I；In single strand dna I, 3 ' ends have by n
The combined area A of a nucleotide composition.
Antibody capture element is made of single strand dna II and two antigen molecules：Single strand dna II is located at centre,
Two ends of single strand dna II are separately connected an antigen molecule corresponding with purpose antibody；In single strand dna II,
There is the combined area B being made of n nucleotide, 3 ' ends to have the combined area C being made of n nucleotide at 5 ' ends；Combined area B and knot
Conjunction area C is reverse sequence；Combined area B and combined area A is reverse complementary sequence.
The principle and flow for the antibody detection method that the present invention establishes are as follows：
1, probe and antibody capture element are incubated altogether, form DNA molecular beacon (combined area A, the combined area B of three chain of part
Form three chains with combined area C, by Waston-Crick Hydrogenbonds between wherein combined area A and combined area B, combined area A and
Combined area C and between pass through Hoogsteen Hydrogenbonds)；
2, sample to be tested is added：If containing purposeful antibody, the decohesion DNA molecular of antibody and antigen in sample to be tested
The structure of beacon, probe are released (Fig. 1 a), and probe generates characteristic signal (Fig. 1 f) by nano-pore single channel, further
The antibody in sample to be tested can be quantified by the frequency that characteristic signal occurs；If without purposeful anti-in sample to be tested
Body, then probe cannot be released, do not generate characteristic signal.
Molecular beacon Detection of Stability method in the present invention is as follows：Testing molecule beacon is added to embodiment 2 and is prepared
Sample cell cis compartments, drawing solution about 15 times, solution is uniformly mixed repeatedly, obtains system to be measured, three chain of test antibodies
A concentration of 200nM of the DNA molecular beacon in system to be measured.Under the conditions of 25 ± 2 DEG C of room temperature letter is carried out using patch clamp amplifier
Number acquisition, and is converted to digital signal by digital analog converter by current signal.Signal sampling frequencies：100kHz, Bessel low pass
Filtering is by frequency：10kHz.The analysis of patch-clamp current signal is analyzed using 10.2 softwares of Clampfit, characteristic signal hand
Data are handled and are fitted using Origin 8.5 by dynamic extraction and manual measurement.This method test molecule beacon is due to this
Signal frequency caused by the probe that body structure is unstable and releases, the signal frequency the high, illustrates that structure is more unstable.Point
The antigen molecule used in the experiment of sub- beacon Detection of Stability for embodiment 3 in the corresponding antigen molecule of five kinds of test antibodies (have
Body is referring to embodiment 3), the optimal molecular beacon that stability experiment filters out in summary screen to obtain by result.
The preparation of embodiment 1, DNA molecular beacon
One, the preparation of antibody capture element
1, single strand dna II is prepared
Artificial synthesized each single strand dna II respectively.
7 kinds of single strand dna II are designed altogether, it is as follows respectively：
(1)
(2)
(3)
(4)
(5)
(6)
(7)
Single underscore marks combined area B, and double underline marks combined area C, is middle area between combined area B and combined area C
Section.
2, the 5 ' ends and 3 ' ends of the DNA molecular synthesized in step (1) respectively connect an antigen molecule (by raw work biology
Antigen molecule is simultaneously connected to DNA molecular end by the direct synthetic antigen molecule of company by covalent bond), it is numbered (1)-(7)
Antibody capture element.
Two, the preparation of probe
1, the artificial synthesized each single strand dna I of difference, at the same it is alkynyl-modified in 5 ' ends progress phosphate group.
3 kinds of single strand dna I are designed altogether, it is as follows respectively：
(a)5’-CCCCCCCCCCCCGAGAGAGA- 3 ' (sequences 2 of sequence table)；
(b)5’-CCCCCCCCCCCCGAGAGA-3’；
(c)5’-CCCCCCCCCCCCGAGAGAGAGA-3’：
Underscore indicates combined area A.
2, take 3.3 μ L single strand dna I solution (solvent is water, a concentration of the 100 of single strand dna I prepared by step 1
μM), 1.2 μ L deionized waters, 2 μ L nitrine solution of ferrocene (solvent is acetonitrile, a concentration of 200mM of nitrine ferrocene), 1 μ L are anti-
Bad hematic acid sodium solution (solvent is water, a concentration of 20mM of sodium ascorbate), (solvent is water, copper nitrate to 0.5 μ L copper nitrate solutions
A concentration of 20mM), the mixing of 2 μ L HEPES buffer solutions (200mM), mixed solution totally 10 μ L.
3, the mixed solution for obtaining step 2 is vortexed at room temperature reacts 2h, and 2 μ L EDTA solution (100mM) are then added eventually
Only react.
4, after completing step 3, using 6 gel exclusion chromatography columns of Micro bio-spin, by specification operation is removed
Salt.
5,10 μ L melons rings { Q [7] } (5mM) are added in the sample obtained after step 4 desalination and are incubated 2h, it is (a)-to obtain number
(c) probe.
Probe that number is (a)-(c) prepare after the completion of structure include single strand dna I, phosphate group, triazole,
Ferrocene and melon ring { Q [7] }；The end modified phosphate group alkynyl of DNA molecular chain 5 ' and nitrine generation click chemistry are anti-
It answers and modifies ferrocene molecule, and then the probe that host-guest interaction forms DNA- ferrocene-melon ring occurs with melon ring, DNA divides
Son is keyed with ferrocene by chemistry, and ferrocene is connect with melon ring { Q [7] } by Subjective and Objective supermolecular mechanism.
Three, the composition design of DNA molecular beacon
It is by the number that the antibody capture element for number (1)-(7) that step 1 obtains is obtained with step 2 respectively successively
(a) probe of-(c) carries out various combination, obtains following DNA molecular beacon：
I：The antibody capture element for numbering (1) is mixed with the probe of number (a) according to equimolar amounts, it is common at 4 DEG C
It is incubated 2h, forms DNA molecular beacon I.
II：The antibody capture element for numbering (2) is mixed with the probe of number (a) according to equimolar amounts, it is common at 4 DEG C
It is incubated 2h, forms DNA molecular beacon II.
III：The antibody capture element for numbering (3) is mixed with the probe of number (a) according to equimolar amounts, at 4 DEG C altogether
With 2h is incubated, DNA molecular beacon III is formed.
IV：The antibody capture element for numbering (4) is mixed with the probe of number (a) according to equimolar amounts, it is common at 4 DEG C
It is incubated 2h, forms DNA molecular beacon IV.
V：The antibody capture element for numbering (5) is mixed with the probe of number (a) according to equimolar amounts, it is common at 4 DEG C
It is incubated 2h, forms DNA molecular beacon V.
VI：The antibody capture element for numbering (6) is mixed with the probe of number (b) according to equimolar amounts, it is common at 4 DEG C
It is incubated 2h, forms DNA molecular beacon VI.
VII：The antibody capture element for numbering (7) is mixed with the probe of number (c) according to equimolar amounts, at 4 DEG C altogether
With 2h is incubated, DNA molecular beacon VI is formed.
VIII：The antibody capture element for numbering (1) is mixed with the probe of number (c) according to equimolar amounts, at 4 DEG C altogether
With 2h is incubated, DNA molecular beacon VI is formed.
The stability of DNA molecular beacon I-VIII is detected and to carry out analysis result as follows：
1) stability of DNA molecular beacon I, DNA molecular beacon II and DNA molecular beacon III are compared.As a result it shows
Show, the centre portion of the antibody capture element of DNA molecular beacon II is 21 base A, the antibody capture of DNA molecular beacon III
The centre portion of element is 21 base C, and the DNA molecular beacon stability that the two is formed is not so good as DNA molecular beacon I (antibody
The centre portion of capture element is 21 base T), therefore, centre portion selection base T is more advantageous to detection and analysis (Fig. 1 b).
2) stability of DNA molecular beacon I, DNA molecular beacon IV and DNA molecular beacon V are compared.DNA molecular
The centre portion of the antibody capture element of beacon IV is 8 bases, and three stranded DNA structure is unstable；The antibody of DNA molecular beacon V
The centre portion of capture element is 21 bases, although structure is more stable, is not easy to release probe point after determinand is added
Son and be unfavorable for detecting；The centre portion of the antibody capture element of DNA molecular beacon I is 21 bases, both can guarantee triple strand dna
Structure is sufficiently stable, is easier to release probe molecule after determinand is added, therefore is conducive to test and analyze (Fig. 1 c).
3) stability of DNA molecular beacon I, DNA molecular beacon VI and DNA molecular beacon VII are compared.As a result it shows
Show, the combined area B of the antibody capture element of DNA molecular beacon VI only contains 3 pairs of TC bases, and the three stranded DNA structure of formation is unstable
It is fixed, cause background interference higher；The combined area B of the antibody capture element of DNA molecular beacon VII contains 5 pairs of TC bases, although shape
At three stranded DNA structure stablize, but its structure again excessively stablize, that is, be added determinand after be not easy to release probe molecule,
Therefore it is unfavorable for detecting.The combined area B of the antibody capture element of DNA molecular beacon I contains 4 pairs of TC bases, both can guarantee three chains
DNA structure is sufficiently stable, is easier to release probe molecule after determinand is added, therefore is conducive to detection and analysis (figure
1d)。
4) stability of DNA molecular beacon I and DNA molecular beacon VIII are compared.The spy of DNA molecular beacon VIII
Needle suspends several bases (combination section B base of the combined area A base numbers of probe more than antibody capture element in midair in combined area A
Number), and the probe of DNA molecular beacon I combine section sealing (the combined area A base numbers of probe be equal to antibody capture member
The combined area B base numbers of part), the stability of DNA molecular beacon I is more preferable (Fig. 1 e).
The above results show that triple strand dna molecular beacon I is best for subsequent detection analytical effect.
The structure of embodiment 2, sample cell system
Sample cell system includes two compartments (cis and trans), and the volume of two compartments is 1.5mL, and two compartments are adopted
(there are one the apertures that diameter is about 100-150 μM among film) is separated with the polytetrafluoroethylene film of 20 μ m-thicks, uses glass fiber
The mixed solution that 1 parts by volume hexadecane and 10 parts by volume pentanes form is instilled the aperture week of polytetrafluoroethylene film by tubule
(dripping quantity is about 2 to 3 microlitres) is enclosed, electrolyte buffer liquid (3M KCl, 10mM Tris, pH then will be added in two compartments
5.0) (1,2- bis- phytane acyl lecithin is added in the form of phosphatide stoste, phosphatide with 1,2-, bis- phytane acyl lecithin (lipids)
A concentration of 10 mg/ml of stoste, addition are about 15 microlitres；The addition of electrolyte buffer liquid is 1mL), liquid relief is used in combination
Rifle mixing, makes it be self-assembly of phospholipid bilayer.Then it is poly- that 1 microlitre of alpha hemolysin seven is added into the solution of cis compartments
Body protein solution (albumen concentration is about 0.3 mg/ml), the spontaneous insertion phospholipid bilayer of albumen form nano-pore, application
Ag/AgCl electrodes (being connected with high frequency sampling probe, high frequency sampling probe is connected with patch clamp amplifier) are in sample cell system
Form closed circuit.
The preparation method of above-mentioned alpha hemolysin heptamer protein solution is as follows：
1, wild type alpha hemolysin plasmid (WT-D8H6)：Bibliography：Bhakdi, S.；Fiissle, R.；Tranum-
Jensen, J.Proc.Natl.Acad.Sci.U.S.A.1981,78,5475-5479.；The public can be from Chinese Academy of Sciences's chemistry
Research institute obtains.
2, the wild type alpha hemolysin plasmid (WT-D8H6) in step 1 is imported into (E.coli in E. coli system
BL21 (DE3) pLysS strain) (bibliography Bhakdi, S.；Fiissle, R.；Tranum-Jensen,
J.Proc.Natl.Acad.Sci.U.S.A.1981,78,5475-5479.) it is expressed, expression is utilized into SDS denaturation
8% polyacrylamide gel electrophoresis obtains monomer after purification, and monomer is added into rabbit erythrocyte later, and monomer will be spontaneous poly-
Synthesize heptamer；Finally albumen is purified, is purified with 8%SDS-PAGE denaturation glue, obtains wild type after purification
(the specific preparation method of alpha hemolysin heptamer protein solution can refer to document to alpha hemolysin heptamer protein solution：Zhuo Lixia,
Wang Ying, Zhang Chunping wait the expression of alpha hemolysins and its preparation [J] Chinese biological engineering magazines of nano-pore, 2017,37 (1)：
53-57.)。
The detection of embodiment 3, antibody
Test antibodies：Dig antibody (Abcam companies, article No.：Ab6212), 5BrdU antibody (Abcam companies, article No.：
Ab8152), HIVp17 antibody (Abcam companies, article No.：Ab9067), Streptavidin (Sigma-Aldrich companies, article No.：
And dig fab fragement (Roche companies, article No. 85878)：11093274910).
One, the preparation and antibody test of three chain molecular beacons
5 ' and 3 ' ends of 1, DNA molecular shown in artificial synthesized sequence 1, the DNA molecular shown in sequence 1 use simultaneously
The corresponding antigen molecule of Dig antibody (digoxin molecule) is modified, Dig antibody capture elements；By Dig antibody captures element with
Number is that the probe of (a) is mixed according to equimolar amounts in embodiment 1, is incubated 2h jointly at 4 DEG C, forms Dig antibody dna molecules
Beacon.
Shown in Dig antibody captures element such as formula (1).
5 ' and 3 ' ends of 2, DNA molecular shown in artificial synthesized sequence 1, the DNA molecular shown in sequence 1 use simultaneously
The corresponding antigen molecule of 5BrdU antibody (5BrdU bases) is modified, 5BrdU antibody capture elements；By 5BrdU antibody captures
Element is that the probe of (a) is mixed according to equimolar amounts with number in embodiment 1, is incubated 2h jointly at 4 DEG C, it is anti-to form 5BrdU
Body DNA molecular beacon.
Shown in 5BrdU antibody captures element such as formula (2).
5 ' and 3 ' ends of 3, DNA molecular shown in artificial synthesized sequence 1, the DNA molecular shown in sequence 1 use simultaneously
HIV p17 antibody corresponding antigen molecule (p17 polypeptides：The ends N-terminal-ELDRWEKIRLRP-C) it is modified, HIV p17 antibody is caught
Obtain element；With number in embodiment 1 it is that the probe of (a) is mixed according to equimolar amounts by HIV p17 antibody captures elements, at 4 DEG C
Lower common incubation 2h, forms HIV p17 antibody dna molecular beacons.
In the antibody capture molecule, the 5 ' ends and 3 ' ends of DNA molecular are connect with p17 polypeptides respectively.
5 ' and 3 ' ends of 4, DNA molecular shown in artificial synthesized sequence 1, the DNA molecular shown in sequence 1 use simultaneously
The corresponding antigen molecule of Streptavidin antibody (biotin molecule) is modified, Streptavidin antibody capture element；By chain
Mould Avidin antibody capture element is that the probe of (a) is mixed according to equimolar amounts with number in embodiment 1, is incubated jointly at 4 DEG C
2h is educated, Streptavidin antibody dna molecular beacon is formed.
Shown in Streptavidin antibody capture element such as formula (3).
5 ' and 3 ' ends of 5, DNA molecular shown in artificial synthesized sequence 1, the DNA molecular shown in sequence 1 use simultaneously
The corresponding antigen molecule of dig fab fragement antibody (digoxin molecule) is modified, dig fab fragement antibody
Capture element；It is the probe of (a) according to equimolar by number in dig fab fragement antibody captures elements and embodiment 1
Amount mixing, is incubated 2h jointly at 4 DEG C, forms dig fab fragement antibody dna molecular beacons.
Shown in dig fab fragement antibody captures element such as formula (1).
6, the test antibodies of various concentration and test antibodies DNA molecular beacon are mixed, obtains mixed solution (mixed solution
Volume be no more than 60 microlitres), by 25 DEG C of mixed solution incubation 30min, mixed solution is added to the sample prepared to embodiment 2
The cis compartments in pond, drawing solution about 15 times, solution is uniformly mixed repeatedly, obtains system to be measured, test antibodies are in system to be measured
In concentration be respectively 0nM, 0.5nM, 10nM, 30nM, 50nM and 100nM, test antibodies DNA molecular beacon is in system to be measured
A concentration of 200nM.
7, after completing step 6, signal acquisition is carried out using patch clamp amplifier under the conditions of 25 ± 2 DEG C of room temperature, and by digital-to-analogue
Current signal is converted to digital signal by converter.
Signal sampling frequencies：100kHz, Bessel low-pass filtering are by frequency：10kHz.
The analysis of patch-clamp current signal is analyzed using 10.2 softwares of Clampfit, characteristic signal manual extraction and hand
It is dynamic to measure, data are handled and are fitted using Origin 8.5.
The testing result of five kinds of test antibodies is respectively as shown in Fig. 2-Fig. 6.The detection limit of five kinds of antibody is respectively dig：
0.5nM、5BrdU：0.8nM、HIV p17：0.5nM, Streptavidin：8nM、dig fab fragement：5nM.
Two, antibody is selectively tested
1, dig antibody selective enumeration method is tested
Test antibodies：Dig antibody (Abcam companies, article No.：Ab6212), 5BrdU antibody (Abcam companies, article No.：
Ab8152), HIV p17 antibody (Abcam companies, article No.：ab9067).
Test antibodies and Dig antibody dna molecular beacons are mixed, obtaining mixed solution, (volume of mixed solution is no more than
60 microlitres), by 25 DEG C of incubation 30min of mixed solution, mixed solution is added to the cis compartments of the sample cell prepared to embodiment 2,
Drawing solution about 15 times repeatedly, solution is uniformly mixed, and obtains system to be measured, and test antibodies are a concentration of in system to be measured
100nM, a concentration of 200nM of the Dig antibody dnas molecular beacon to be measured in system to be measured.The control group for not adding antibody is set.
Signal acquisition is carried out using patch clamp amplifier under the conditions of 25 ± 2 DEG C of room temperature, and is believed electric current by digital analog converter
Number be converted to digital signal.
Signal sampling frequencies：100kHz, Bessel low-pass filtering are by frequency：10kHz.
The analysis of patch-clamp current signal is analyzed using 10.2 softwares of Clampfit, characteristic signal manual extraction and hand
It is dynamic to measure, data are handled and are fitted using Origin 8.5.
The results are shown in Figure 7.Interfere antibody (5BrdU antibody, HIVp17 antibody) minimum to the interference of this experiment.
2,5BrdU antibody selective enumeration method is tested
Test antibodies：Test antibodies：Dig antibody (Abcam companies, article No.：Ab6212), 5BrdU antibody (Abcam companies,
Article No.：Ab8152), HIVp17 antibody (Abcam companies, article No.：ab9067).
Test antibodies and 5BrdU antibody dna molecular beacons are mixed, obtaining mixed solution, (volume of mixed solution does not surpass
Cross 60 microlitres), by 25 DEG C of mixed solution incubation 30min, by mixed solution be added the cis of the sample cell prepared to embodiment 2 every
Room, drawing solution about 15 times, solution is uniformly mixed repeatedly, obtains system to be measured, concentration of the test antibodies in system to be measured
For 100nM, a concentration of 200nM of the Dig antibody dnas molecular beacon to be measured in system to be measured.The control for not adding antibody is set
Group.
Signal acquisition is carried out using patch clamp amplifier under the conditions of 25 ± 2 DEG C of room temperature, and is believed electric current by digital analog converter
Number be converted to digital signal.
Signal sampling frequencies：100kHz, Bessel low-pass filtering are by frequency：10kHz.
The analysis of patch-clamp current signal is analyzed using 10.2 softwares of Clampfit, characteristic signal manual extraction and hand
It is dynamic to measure, data are handled and are fitted using Origin 8.5.
The results are shown in Figure 8.Interfere antibody (Dig antibody, HIV p17 antibody) minimum to the interference of this experiment.
3, HIV p17 antibody selective enumeration method is tested
Test antibodies：Test antibodies：Dig antibody (Abcam companies, article No.：Ab6212), 5BrdU antibody (Abcam companies,
Article No.：Ab8152), HIVp17 antibody (Abcam companies, article No.：ab9067).
Test antibodies and HIV p17 antibody dna molecular beacons are mixed, obtaining mixed solution, (volume of mixed solution is not
More than 60 microlitres), by 25 DEG C of incubation 30min of mixed solution, mixed solution is added to the cis of the sample cell prepared to embodiment 2
Compartment, drawing solution about 15 times, solution is uniformly mixed repeatedly, obtains system to be measured, and test antibodies are dense in system to be measured
Degree is 100nM, a concentration of 200nM of the Dig antibody dnas molecular beacon to be measured in system to be measured.Pair for not adding antibody is set
According to group.
Signal acquisition is carried out using patch clamp amplifier under the conditions of 25 ± 2 DEG C of room temperature, and is believed electric current by digital analog converter
Number be converted to digital signal.
Signal sampling frequencies：100kHz, Bessel low-pass filtering are by frequency：10kHz.
The analysis of patch-clamp current signal is analyzed using 10.2 softwares of Clampfit, characteristic signal manual extraction and hand
It is dynamic to measure, data are handled and are fitted using Origin 8.5.
The results are shown in Figure 9.Interfere antibody (Dig antibody, 5BrdU antibody) minimum to the interference of this experiment.
<110>Institute of Chemistry, Academia Sinica
Institute of High Energy Physcis, Academia Sinica
<120>Antibody detection method associated with a kind of triple strand dna molecular beacon combination nano-pore technology
<160> 2
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
cccccccccc ccgagagaga 20
<210> 2
<211> 37
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
tctctctctt tttttttttt tttttttttc tctctct 37
Claims (10)
1. a kind of molecular combinations for detecting target antibody, including probe molecule and antibody capture molecule；
The probe molecule is to modify melon ring to obtain in the 5 ' ends of single strand dna I；In single strand dna I, 3 ' ends
With the combined area A being made of n nucleotide；
The antibody capture molecule is made of single strand dna II and two antigen molecules；Single strand dna II is located at centre,
Two ends of single strand dna II are separately connected an antigen molecule corresponding with purpose antibody；In DNA molecular II, 5 ' ends
With the combined area B being made of n nucleotide, 3 ' ends have the combined area C being made of n nucleotide；
Combined area B and combined area C is reverse sequence；Combined area B and combined area A is reverse complementary sequence.
2. a kind of compound for detecting target antibody is incubated is formed altogether by probe molecule and antibody capture molecule；
The probe molecule is to modify melon ring to obtain in the 5 ' ends of single strand dna I；In single strand dna I, 3 ' ends
With the combined area A being made of n nucleotide；
The antibody capture molecule is made of single strand dna II and two antigen molecules；Single strand dna II is located at centre,
Two ends of single strand dna II are separately connected an antigen molecule corresponding with purpose antibody；In DNA molecular II, 5 ' ends
With the combined area B being made of n nucleotide, 3 ' ends have the combined area C being made of n nucleotide；
Combined area B and combined area C is reverse sequence；Combined area B and combined area A is reverse complementary sequence.
3. the compound described in molecular combinations as described in claim 1 or claim 2, it is characterised in that：
Random natural numbers of the n between 6-10；
The length of the single strand dna I is 18-22bp；
The length of the single strand dna II is 33-41bp.
4. the molecular combinations as described in claims 1 to 3 is any, or, compound according to claim 2 or 3, feature exist
In：
The n is 8；
The length of the single strand dna I is 20bp；
The length of the single strand dna II is 37bp.
5. any molecular combinations of Claims 1-4, or, any compound of claim 2 to 4, feature exist
In：The compound that can connect melon ring with DNA molecular is ferrocene.
6. any molecular combinations of claim 1 to 5, or, any compound of claim 2 to 5, feature exist
In：The single strand dna I is as shown in the sequence 1 of sequence table；The single strand dna II is as shown in the sequence 2 of sequence table.
7. a kind of system, including claim 1 to the 6 any molecular combinations and sample cell system；
The sample cell system includes two compartments, and two compartments are separated by polytetrafluoroethylene film；In the polytetrafluoroethylene film
Between tool there are one a diameter of 100-150 μM of holes, the through-hole is full of by phospholipid bilayer, in the phospholipid bilayer
The nano-pore formed by alpha hemolysin heptamer albumen there are one；It is closed using Ag/AgCl electrodes composition in sample cell system
Circuit.
8. a kind of system, including claim 2 to the 6 any compound and sample cell system；
The sample cell system includes two compartments, and two compartments are separated by polytetrafluoroethylene film；In the polytetrafluoroethylene film
Between tool there are one a diameter of 100-150 μM of holes, the through-hole is full of by phospholipid bilayer, in the phospholipid bilayer
The nano-pore formed by alpha hemolysin heptamer albumen there are one；It is closed using Ag/AgCl electrodes composition in sample cell system
Circuit.
9. any molecular combinations of claim 1 to 6, or, any compound of claim 2 to 6, or, right
It is required that application of the system in testing goal antibody described in 7 or 8.
10. the method for purpose antibody, includes the following steps in a kind of detection sample：Sample to be tested is any with claim 2 to 6
The compound is added after being incubated jointly in the sample cell system described in claim 7 or 8, special by analysing whether to generate
Levy detection of the electric signal realization to purpose antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810198020.2A CN108467883A (en) | 2018-03-09 | 2018-03-09 | Antibody detection method associated with a kind of triple strand dna molecular beacon combination nano-pore technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810198020.2A CN108467883A (en) | 2018-03-09 | 2018-03-09 | Antibody detection method associated with a kind of triple strand dna molecular beacon combination nano-pore technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108467883A true CN108467883A (en) | 2018-08-31 |
Family
ID=63265242
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810198020.2A Pending CN108467883A (en) | 2018-03-09 | 2018-03-09 | Antibody detection method associated with a kind of triple strand dna molecular beacon combination nano-pore technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108467883A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607349A (en) * | 2019-08-27 | 2019-12-24 | 中国科学院化学研究所 | Enzyme activity concentration detection method combining host-guest probe and nanopore technology |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103983487A (en) * | 2014-04-11 | 2014-08-13 | 西北大学 | Method for assembling alpha-hemolysin (alphaHL) protein nanopores by using single-layer phospholipid membrane |
-
2018
- 2018-03-09 CN CN201810198020.2A patent/CN108467883A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103983487A (en) * | 2014-04-11 | 2014-08-13 | 西北大学 | Method for assembling alpha-hemolysin (alphaHL) protein nanopores by using single-layer phospholipid membrane |
Non-Patent Citations (5)
| Title |
|---|
| BINGYUAN GUO，HAICHEN WU: "Analyte-Triggered DNA‐Probe Release from a Triplex Molecular Beacon for Nanopore Sensing", 《ANGEWANDTE CHEMIE》 * |
| SIMONA RANALLO, FRANCESCO RICCI ET AL.,: "A Modular, DNA‐Based Beacon for Single‐Step Fluorescence Detection of Antibodies and Other Protein", 《ANGEWANDTE CHEMIE》 * |
| SIMONA RANALLO, FRANCESCO RICCI ET AL.,: "Antibody-powered nucleic acid release using a DNA-based nanomachine", 《NATURE COMMUNICATIONS》 * |
| TING LI, HAI-CHEN WU等: "A Universal Strategy for Aptamer‐Based Nanopore Sensing through Host–Guest Interactions inside α‐Hemolysin", 《ANGEWANDTE CHEMIE INT. ED.》 * |
| 卓丽霞等，: "α-溶血素的表达及其纳米孔的制备", 《中国生物工程杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607349A (en) * | 2019-08-27 | 2019-12-24 | 中国科学院化学研究所 | Enzyme activity concentration detection method combining host-guest probe and nanopore technology |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20230392157A1 (en) | Aptamer method | |
| Kraly et al. | Microfluidic applications in metabolomics and metabolic profiling | |
| US20180335418A1 (en) | Multiplexed biomarker quantitation by nanopore analysis of biomarker-polymer complexes | |
| JP2002506200A (en) | Biosensor array containing cell populations confined in micropores | |
| CN105259229B (en) | A kind of single molecule analysis method for detecting medicine | |
| CN109142710B (en) | Method for rapidly and sensitively detecting tetrodotoxin TTX | |
| Li et al. | Designing of molecularly imprinted polymer-based potentiometric sensor for the determination of heparin | |
| Liang et al. | Competitive method-based electrochemiluminescent assay with protein–nucleotide conversion for ratio detection to efficiently monitor the drug resistance of cancer cells | |
| CN106929565A (en) | Protein monomolecular electronic device and its preparation and application based on nanostructured | |
| WO2006126570A1 (en) | Method for simultaneous analysis of multiple bioilogical reactions or changes in in vivo conditions | |
| CN106471369A (en) | Target detection using nano-pores | |
| US20080156646A1 (en) | Nanostructured electrochemical biosensor with aptamer as molecular recognition probe | |
| Shi et al. | Signal amplification in capillary electrophoresis based chemiluminescent immunoassays by using an antibody–gold nanoparticle–DNAzyme assembly | |
| Huang et al. | Solid-state nanochannels for bio-marker analysis | |
| Yan et al. | DNA aptamer folding on magnetic beads for sequential detection of adenosine and cocaine by substrate-resolved chemiluminescence technology | |
| CN108841828B (en) | Single-stranded DNA aptamer for specifically recognizing tobramycin and application thereof | |
| WO2001046693A2 (en) | Screening under isothermal denaturing conditions | |
| CN108467883A (en) | Antibody detection method associated with a kind of triple strand dna molecular beacon combination nano-pore technology | |
| CN111455034B (en) | Single molecule detection method and system based on solid-state nanopore mechanism | |
| US20010036626A1 (en) | Screening assay methods and systems using target pooling | |
| CN115436335B (en) | Method for detecting thrombin based on perylene derivative probe without marking | |
| CN112611876B (en) | Urine amino acid detection kit and method for aptamer-quadruplex | |
| CN104048953B (en) | The method for quick of a kind of trace levels of endotoxin and kit | |
| Yu et al. | A colorimetric biosensor for ultrasensitive detection of the SURF1 gene based on a dual DNA-induced cascade hybridization reaction | |
| DE60029363T2 (en) | Fluorescence method for measuring enzyme activity using polyions |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180831 |
|
| RJ01 | Rejection of invention patent application after publication |