CN107760759A - The method for detecting prostate cancer target methyl amimoacetic acid - Google Patents

The method for detecting prostate cancer target methyl amimoacetic acid Download PDF

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CN107760759A
CN107760759A CN201711085501.4A CN201711085501A CN107760759A CN 107760759 A CN107760759 A CN 107760759A CN 201711085501 A CN201711085501 A CN 201711085501A CN 107760759 A CN107760759 A CN 107760759A
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dna
origami
hrp
sox
methyl amimoacetic
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何丹农
徐艳
陈玮嘉
王萍
金彩虹
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Shanghai National Engineering Research Center for Nanotechnology Co Ltd
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    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/342—Prostate diseases, e.g. BPH, prostatitis

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Abstract

The present invention provides a kind of method for detecting prostate cancer target methyl amimoacetic acid, detected respectively with two kinds of DNA paper foldings assemblings, SOx origami compounds III and the assembling of HRP origami compounds IV and methyl amimoacetic acid with DNA assemblings, the synthesis of tubulose DNA paper foldings, enzyme dna compound respectively including SOx and HRP, cascade enzyme SOx is connected with tubulose DNA paper foldings of the HRP respectively with two kinds, the orderly cascade between two enzymes is realized using the strong base interaction of the complementary arm chain of two paper foldings stretchings, reaches the purpose for improving detection sensitivity.The new detection method that can be achieved to regulate and control SOx and HRP spacing in order is established using tubulose origami structure.By the way of further being assembled after enzyme and paper folding assemble respectively, improve because of the packaging efficiency difference caused by space steric effect the defects of, improve the packaging efficiency of two enzymes.

Description

The method for detecting prostate cancer target methyl amimoacetic acid
Technical field
A kind of method for detecting prostate cancer target methyl amimoacetic acid of present invention design, and in particular to utilize DNA tubulose paper folding knots Structure builds the detection architecture based on cascade reaction, and content of sarcosine is measured by chromogenic reaction.The invention belongs to biology Sensing detection field.
Background technology
With the aggravation of Chinese society's aging, explosive growth is presented in prostate cancer at home.Due to benign prostatic Cancer have can radical-ability, existing early screening is mainly for prostate specific antigen（PSA）Carry out, its detection sensitivity and spy There is larger problem in the opposite sex, false positive rate is higher.And there is an urgent need to develop to be directed to other for the prostate-cancer incidence constantly risen Prostate cancer target object detection method.Metabolism group research shows methyl amimoacetic acid（Sarcosine）Prostate cancer development process is participated in, It is the potentiality target of prostate cancer early screening and content significantly raises.
Sarcosine oxidase（SOx）The hydrogen peroxide for being catalyzed methyl amimoacetic acid production can be further by horseradish peroxidase （HRP）Using catalytic chemistry chromogenic reaction, this group of cascade reaction can be employed the measure of content of sarcosine.Due to solution middle reaches From cascade enzyme be in unordered state, its catalytic efficiency is to a certain extent by the random collision of substrate and enzyme.Study table The spacing of bright strict control cascade enzyme can regulate and control the catalytic activity of enzyme, significantly improve the efficiency of cascade reaction.In various structures In the method for cascade reaction, DNA nanostructure shows its exclusive advantage, special DNA paper folding arts（DNA origami）Go out Now make it possible the control of complex three-dimensional structure.DNA paper foldings have easily modification, high-biocompatibility and space addressable excellent Gesture, application prospect are extensive.
The content of the invention
The problem of false positive rate is high in being detected for PSA, present invention aims at：Detect prostate cancer target methyl amimoacetic acid Method.
The present invention is directed to the problem of positive rate of prostate cancer early screening, and PSA is replaced as forefront using methyl amimoacetic acid Gland cancer target, using DNA paper folding ordered arrangement cascade reactions, so as to build a kind of method of prostate cancer target detection, including Following steps：
（1）Sarcosine oxidase（SOx）With horseradish peroxidase（HRP）Assembled respectively with DNA：By SOx, HRP respectively with connecting Connect agent 3- (2- pyridines dimercapto) propionic acid N-hydroxy-succinamide ester（SPDP）After 25 DEG C of 2 hr of incubation being mixed with certain proportion, Ultrafiltration washes away excessive SPDP；Two kinds of enzymes of subsequent SPDP modifications respectively the DNA 1 with sulfydryl modification, DNA 2 with certain proportion Uniform 25 DEG C of overnight incubations, wash away excessive DNA using ultrafiltration afterwards, obtain SOx-DNA complexⅠs and HRP-DNA compound Ⅱ；
（2）The synthesis of tubulose DNA paper foldings：Equal proportion is well mixed to replace chain DNA and original DNA staples chain, in 1 × TAE- Mg2+In solution, staple chain is well mixed after annealing with certain proportion with long chain DNA and forms DNA origami structures；Using ultrafiltration Excessive staple chain is washed away, DNA origami I and DNA origami II are respectively obtained according to chain difference is replaced；
（3）Enzyme-DNA compounds assemble with two kinds of DNA paper foldings respectively：SOx-DNA complexⅠs (or HRP-DNA complexⅱs) with Origami I or origami II are well mixed with certain proportion, in 45 DEG C of annealing assemblings；Separated by agarose gel electrophoresis The enzyme to dissociate in annealed product and paper folding are removed, is obtained containing SOx-origami compounds III and HRP-origami compounds IV Band, removing glue, recovery SOx-origami compounds III and HRP-origami compounds IV are purified using Filter column；
（4）The assembling of SOx-origami compounds III and HRP-origami compounds IV：By the He of SOx-origami compounds III HRP-origami compounds IV are well mixed with certain proportion, and in 37 DEG C of annealing assemblings, it is compound to obtain SOx-origami-HRP Thing V；
（5）Methyl amimoacetic acid detects：The reaction mother liquor of methyl amimoacetic acid and HRP catalyzed coloration substrates is configured in advance, by finite concentration during detection Methyl amimoacetic acid and chromogenic substrate are well mixed, and are added the SOx-origami-HRP compounds V of certain content, are selected according to chromogenic substrate Suitable instrument is selected to be measured.
The present invention is proposed using methyl amimoacetic acid as prostate cancer target, utilizes inspection of the DNA origami structures structure based on cascade enzyme Survey system, improve detection sensitivity.
It is that cascade enzyme SOx is connected with two kinds of tubulose DNA paper foldings respectively with HRP, utilizes two paper foldings in the principle of the invention The strong base of the arm interchain of stretching mutually realizes the orderly cascade between two enzymes, reaches the purpose for improving detection sensitivity.
On the basis of such scheme, described buffer solution is excellent when being this area routine cushioning liquid, enzyme modification DNA and detection The buffering of choosing is molten for pH=7.4,8 mM Na2HPO4、2 mM KH2PO4, 136 mM Na Cl, 2.6 mM K Cl, 1 × PBS delay Fliud flushing；The buffer solution of DNA paper foldings synthesis and agarose gel electrophoresis be pH=8.0,40 mM Tris- acetic acid, 1 mM EDTA, 12.5 mM MgCl2 1×TAE-Mg2+Solution.
Described enzyme and DNA assembling ratio can be the conventional ratio in this area, and preferable ratio is 1:10；Described DNA paper foldings can be the conventional different size of tubulose DNA paper foldings in this area, preferable a width of 6 nm of tubulose DNA paper foldings, the length of side For 412 nm；Described long-chain single stranded DNA can be the conventional long-chain in this area, preferably M13mp18 single stranded DNAs.
Described preferable paper folding assembling mode comprises the following steps：Single-stranded M13mp18 and the staple chain DNA are mixed Together in 1 × TAE-Mg2+In cushioning liquid, 20 DEG C are annealed to from 90 DEG C in PCR instrument, annealing rate is 0.1 DEG C/10s.
Described DNA paper foldings sequence can be the conventional sequence in this area, and preferably August in 2010 is published in Nano on 3rd The entitled Programmable Periodicity of Quantum Dot of Letter the 9th the 3367-3372 pages of phases of volume 10 S6- in the Supporting Online Information of Arrays with DNA Origami Nanotubes paper The DNA sequence dna of Table S1 described in S8 pages, include Seq. No.1 to 22 DNA, wherein, it is complementary with the Sequences of DNA 1 DNA 3 be the Column 1 of Helix 43 ' terminal extensions sequence；Complementary DNA 4 is with the Sequences of DNA 2 The sequence of the Column 1 of Helix 43 ' terminal extensions；The sequence DNA 5-13 replaced in addition is respectively the Column of Helix 1 The sequence of 5/14/23/32/38/47/56/65/74 3 ' terminal extensions；The sequence DNA 14-22 of replacement is respectively Helix 6 The sequence of Column 6,/15,/24,/33,/39,/48,/57,/66,/75 3 ' terminal extensions；Wherein DNA 5-13 3 ' end sequences with The sequence of DNA 14-22 3 ' terminal extensions is complementary；
Described SOx-DNA complexⅠs and HRP-DNA complexⅱ assembles with DNA origami I and DNA origami II Mode can be the conventional mode in this area, 20 DEG C are annealed to from 45 DEG C preferably in PCR instrument, annealing rate 0.1 ℃/15 s。
Described SOx-DNA complexⅠs and HRP-DNA complexⅱ and DNA origami I and DNA origami II The ratio of assembling can be the conventional ratio in this area, and preferable ratio is SOx-DNA complexⅠs or HRP-DNA compounds Ⅱ：Origami I or origami II=10:1.
Further, step（3）In, described SOx-origami compounds III and HRP-origami compounds IV it is pure Change mode is the conventional mode in this area, and preferably agarose gel electrophoresis, preferable agarose concentration are 0.5 %, Preferable deposition condition is the min of 80 V voltage ice baths electrophoresis 80；It is compound to SOx-origami compounds III and HRP-origami The way of recycling of the band of thing IV can be the conventional mode in this area, preferably using Freeze N Squeeze spin Columns is purified.
Further, step（4）In, described SOx-origami compounds III and HRP-origami IV assembling ratio For the conventional ratio in this area, preferable ratio is 1:1.Assembling mode can be the conventional assembling mode in this area, preferably Using being annealed to 10 DEG C from 37 DEG C in PCR instrument, annealing rate is 0.1 DEG C/15 s.
Step（5）In, described HRP substrates are the conventional substrate in this area, and preferably 2,2- joins (the 3- second of nitrogen-two Base-benzothiazole -6- sulfonic acid) di-ammonium salts（ABTS）, 3,3', 5,5'- tetramethyl benzidines（TMB）, 10- acetyl group -3,7- bis- Hydroxyl azophenlyene（Amplex Red）.
Further, step（5）In, the concentration of SOx-origami-HRP compounds V is this area during described detection Normal concentration, preferable concentration are 1 nM.
The advantage of the invention is that：
（1）The new detection method that can be achieved to regulate and control SOx and HRP spacing in order is established using tubulose origami structure.
（2）By the way of further being assembled after enzyme and paper folding assemble respectively, improve because space steric effect causes Packaging efficiency difference the defects of, improve two enzymes packaging efficiency.
Embodiment
Technical scheme is further described below by way of specific embodiment.Following embodiment is to this The further explanation of invention, and do not limit the scope of the invention.
Embodiment 1
SOx, HRP assemble with sulfydryl DNA 1 and DNA 2 respectively：100 μl SOx/HRP（2 μM）Respectively with SPDP with 1:10 ratios Example is incubated at room temperature 2 hr in 1 × PBS.30 kD super filter tube centrifuge washings of excessive SPDP（3000g, 10 min）After three times, 1000g surpasses in reverse filter and reclaimed for 10 minutes, ultraviolet quantitative.The SOx/HRP of SPDP- modifications respectively with 10 times of excess Sulfydryl-DNA 1 and DNA 2 is incubated at room temperature overnight in 1 × PBS.By the change for determining light absorption value at 343 nm Carry out quantitative SOx, HRP and DNA connection ratio.Last unnecessary DNA is removed by the method for centrifugal ultrafiltration（3000g, 10 Min, three times）, 1000g, which is surpassed in reverse, filters recovery in 10 minutes, obtains SOx-DNA complexⅠs and HRP-DNA complexⅱs.
The paper folding of DNA tubuloses synthesizes：Will replace chain DNA 3, DNA 5-13 and remaining do not replace staple chain and be well mixed, it is dilute Release to 200 nM, be designated as staple combination chain I.Will replace chain DNA 4, DNA 14-22 and remaining do not replace staple chain mix Uniformly, 200 nM are diluted to, are designated as staple combination chain II.Take 100 μ l（20 nM）Single-stranded M13mp18 DNA respectively with 10 μ l staples combination chains I or II, 10 10 × TAE-Mg of μ l2+Buffer solution (pH 8) and 70 μ l ultra-pure waters are well mixed, Anneal and assemble in PCR.Using 1 × TAE-Mg2+Buffer solution (pH 8) and the μ L super filter tubes centrifuge washings three of 100 kD 500 It is secondary（3000 g are centrifuged）Unnecessary single stranded DNA is removed, 1000g surpasses in reverse the DNA paper foldings I and II after 10 minutes recovery purifyings of filter.
Enzyme is cascaded to assemble with two kinds of DNA paper foldings respectively：By SOx-DNA complexⅠs and HRP-DNA complexⅱs respectively with folding Paper I and II is with concentration ratio 10:1 ratio is in 1 × TAE-Mg2+Mixed in buffer solution, subsequent mixed liquor is in PCR instrument from 45 DEG C 20 DEG C are annealed to, annealing rate is 0.1 DEG C/15 s.Two kinds of mixtures of PCR are obtained in 0.5 % Ago-Gels（Using 1 × TAE-Mg2+Buffer solution）After the min of 80 V voltage ice baths electrophoresis 80, rubber tapping is obtained with cascade enzyme-He of paper folding compound III The band of compound IV, purified using the further degummings of Freeze N Squeeze spin columns, obtain cascading enzyme-folding Paper compound III and compound IV.
Two kinds of DNA paper foldings assembling with cascade enzyme：Cascade enzyme-paper folding compound III and compound are concentrated using super filter tube IV, ultraviolet quantitative concentrations.5 nM are cascaded into the nM compounds IV of enzyme-paper folding compound III and 5 with 1:1 volume ratio is well mixed, 37 DEG C of annealing are down to 10 DEG C and assembled in PCR.PCR mixtures are obtained in 0.5 % Ago-Gels（Using 1 × TAE-Mg2+Buffer solution）After the min of 80 V voltage ice baths electrophoresis 80, rubber tapping recovery band, using Freeze N Squeeze Degumming is further purified in spin columns, obtains the SOx-origami-HRP compounds V that spacing is 10 nm.
Methyl amimoacetic acid detects：100 μM of methyl amimoacetic acids, 100 mM ABTS mother liquors are respectively configured with PBS, ice bath preserves.Open purple Outer spectrophotometer, parameter is set, correct baseline.0 μ L, 0.5 μ L, 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L are taken successively Methyl amimoacetic acid（100 µM）, 1 μ L ABTS（100 mM）Add 88 μ L PBS to be well mixed, be transferred in cuvette, then add 10 μ L SOx-origami-HRP compounds V, after being well mixed, absorbance change at 414 nm is detected, it is dense to draw methyl amimoacetic acid The standard curve of degree.
Embodiment 2
SOx, HRP assemble with sulfydryl DNA 1 and DNA 2 respectively：100 μl SOx/HRP（2 μM）Respectively with SPDP with 1:10 ratios Example is incubated at room temperature 2 hr in 1 × PBS.30 kD super filter tube centrifuge washings of excessive SPDP（3000g, 10 min）After three times, 1000g surpasses in reverse filter and reclaimed for 10 minutes, ultraviolet quantitative.The SOx/HRP of SPDP- modifications respectively with 10 times of excess Sulfydryl-DNA 1 and DNA 2 is incubated at room temperature overnight in 1 × PBS.By the change for determining light absorption value at 343 nm Carry out quantitative SOx, HRP and DNA connection ratio.Last unnecessary DNA is removed by the method for centrifugal ultrafiltration（3000g, 10 Min, three times）, 1000g, which is surpassed in reverse, filters recovery in 10 minutes, obtains SOx-DNA complexⅠs and HRP-DNA complexⅱs.
The paper folding of DNA tubuloses synthesizes：Will replace chain DNA 3, DNA 5-13 and remaining do not replace staple chain and be well mixed, it is dilute Release to 200 nM, be designated as staple combination chain I.Will replace chain DNA 4, DNA 14-22 and remaining do not replace staple chain mix Uniformly, 200 nM are diluted to, are designated as staple combination chain II.Take 100 μ l（20 nM）Single-stranded M13mp18 DNA respectively with 10 μ l staples combination chains I or II, 10 10 × TAE-Mg of μ l2+Buffer solution (pH 8) and 70 μ l ultra-pure waters are well mixed, Anneal and assemble in PCR.Using 1 × TAE-Mg2+Buffer solution (pH 8) and the μ L super filter tubes centrifuge washings three of 100 kD 500 It is secondary（3000 g are centrifuged）Unnecessary single stranded DNA is removed, 1000g surpasses in reverse the DNA paper foldings I and II after 10 minutes recovery purifyings of filter.
Enzyme is cascaded to assemble with two kinds of DNA paper foldings respectively：By SOx-DNA complexⅠs and HRP-DNA complexⅱs respectively with folding Paper I and II is with concentration ratio 10:1 ratio is in 1 × TAE-Mg2+Mixed in buffer solution, subsequent mixed liquor is in PCR instrument from 45 DEG C 20 DEG C are annealed to, annealing rate is 0.1 DEG C/15 s.Two kinds of mixtures of PCR are obtained in 0.5 % Ago-Gels（Using 1 × TAE-Mg2+Buffer solution）After the min of 80 V voltage ice baths electrophoresis 80, rubber tapping is obtained with cascade enzyme-He of paper folding compound III The band of compound IV, purified using the further degummings of Freeze N Squeeze spin columns, obtain cascading enzyme-folding Paper compound III and compound IV.
Two kinds of DNA paper foldings assembling with cascade enzyme：Cascade enzyme-paper folding compound III and compound are concentrated using super filter tube IV, ultraviolet quantitative concentrations.5 nM are cascaded into the nM compounds IV of enzyme-paper folding compound III and 5 with 1:1 volume ratio is well mixed, 37 DEG C of annealing are down to 10 DEG C and assembled in PCR.PCR mixtures are obtained in 0.5 % Ago-Gels（Using 1 × TAE-Mg2+Buffer solution）After the min of 80 V voltage ice baths electrophoresis 80, rubber tapping recovery band, using Freeze N Squeeze Degumming is further purified in spin columns, obtains the SOx-origami-HRP compounds V that spacing is 10 nm.
Methyl amimoacetic acid detects：100 μM of methyl amimoacetic acids, 100 mM TMB mother liquors are respectively configured with PBS, ice bath preserves.Open ultraviolet Spectrophotometer, parameter is set, correct baseline.0 μ L, 0.5 μ L, 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L fleshes are taken successively Propylhomoserin（100 µM）, 1 μ L TMB（100 mM）Add 88 μ L PBS to be well mixed, be transferred in cuvette, then add 10 μ L SOx-origami-HRP compounds V, after being well mixed, absorbance change at 630 nm is detected, draws sarcosine concentrations Standard curve.
Embodiment 3
SOx, HRP assemble with sulfydryl DNA 1 and DNA 2 respectively：100 μl SOx/HRP（2 μM）Respectively with SPDP with 1:10 ratios Example is incubated at room temperature 2 hr in 1 × PBS.30 kD super filter tube centrifuge washings of excessive SPDP（3000g, 10 min）After three times, 1000g surpasses in reverse filter and reclaimed for 10 minutes, ultraviolet quantitative.The SOx/HRP of SPDP- modifications respectively with 10 times of excess Sulfydryl-DNA 1 and DNA 2 is incubated at room temperature overnight in 1 × PBS.By the change for determining light absorption value at 343 nm Carry out quantitative SOx, HRP and DNA connection ratio.Last unnecessary DNA is removed by the method for centrifugal ultrafiltration（3000g, 10 Min, three times）, 1000g, which is surpassed in reverse, filters recovery in 10 minutes, obtains SOx-DNA complexⅠs and HRP-DNA complexⅱs.
The paper folding of DNA tubuloses synthesizes：Will replace chain DNA 3, DNA 5-13 and remaining do not replace staple chain and be well mixed, it is dilute Release to 200 nM, be designated as staple combination chain I.Will replace chain DNA 4, DNA 14-22 and remaining do not replace staple chain mix Uniformly, 200 nM are diluted to, are designated as staple combination chain II.Take 100 μ l（20 nM）Single-stranded M13mp18 DNA respectively with 10 μ l staples combination chains I or II, 10 10 × TAE-Mg of μ l2+Buffer solution (pH 8) and 70 μ l ultra-pure waters are well mixed, Anneal and assemble in PCR.Using 1 × TAE-Mg2+Buffer solution (pH 8) and the μ L super filter tubes centrifuge washings three of 100 kD 500 It is secondary（3000 g are centrifuged）Unnecessary single stranded DNA is removed, 1000g surpasses in reverse the DNA paper foldings I and II after 10 minutes recovery purifyings of filter.
Enzyme is cascaded to assemble with two kinds of DNA paper foldings respectively：By SOx-DNA complexⅠs and HRP-DNA complexⅱs respectively with folding Paper I and II is with concentration ratio 10:1 ratio is in 1 × TAE-Mg2+Mixed in buffer solution, subsequent mixed liquor is in PCR instrument from 45 DEG C 20 DEG C are annealed to, annealing rate is 0.1 DEG C/15 s.Two kinds of mixtures of PCR are obtained in 0.5 % Ago-Gels（Using 1 × TAE-Mg2+Buffer solution）After the min of 80 V voltage ice baths electrophoresis 80, rubber tapping is obtained with cascade enzyme-He of paper folding compound III The band of compound IV, purified using the further degummings of Freeze N Squeeze spin columns, obtain cascading enzyme-folding Paper compound III and compound IV.
Two kinds of DNA paper foldings assembling with cascade enzyme：Cascade enzyme-paper folding compound III and compound are concentrated using super filter tube IV, ultraviolet quantitative concentrations.5 nM are cascaded into the nM compounds IV of enzyme-paper folding compound III and 5 with 1:1 volume ratio is well mixed, 37 DEG C of annealing are down to 10 DEG C and assembled in PCR.PCR mixtures are obtained in 0.5 % Ago-Gels（Using 1 × TAE-Mg2+Buffer solution）After the min of 80 V voltage ice baths electrophoresis 80, rubber tapping recovery band, using Freeze N Squeeze Degumming is further purified in spin columns, obtains the SOx-origami-HRP compounds V that spacing is 10 nm.
Methyl amimoacetic acid detects：100 μM of methyl amimoacetic acids, 100 mM Amplex Red mother liquors are respectively configured with PBS, ice bath preserves. Sepectrophotofluorometer is opened, sets 561 nm to excite, 565-580 nm transmittings, corrects baseline.Take successively 0 μ L, 0.5 μ L, 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L methyl amimoacetic acids（100 µM）, 1 μ L Amplex Red（100 mM）88 μ L PBS are added to mix Close uniformly, be transferred in cuvette, then add 10 μ L SOx-origami-HRP compounds V, after being well mixed, detection 569 nm change in fluorescence, draw the standard curve of sarcosine concentrations.
<110>Shanghai National Engineering Research Center for Nanotechnology Co., Ltd
<120>The method for detecting prostate target methyl amimoacetic acid
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<213>Artificial sequence
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<400>attctaagaa cgcggggcgc gagctgaatt gtatcatcgc cttttaaaaa aaaaaaaaaa aaaaa
<210>19
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>ttccaagaac gggtcggttg taccaaaact cacattaatt gctttaaaaa aaaaaaaaaa aaaaa
<210>20
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>tgttcagcta atgcaagatt caaaagggag ctcgaattcg tatttaaaaa aaaaaaaaaa aaaaa
<210>21
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>gaatcgccat atttggtcat tgcctgaggg ggatgtgctg catttaaaaa aaaaaaaaaa aaaaa
<210>22
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>aaataaggcg ttaacaaata tttaaattgc cagctttccg gctttaaaaa aaaaaaaaaa aaaaa

Claims (10)

  1. A kind of 1. method for detecting prostate cancer target methyl amimoacetic acid, it is characterised in that using methyl amimoacetic acid as prostate cancer target, profit With detection architecture of the DNA origami structures structure based on cascade enzyme, comprise the following steps：
    （1）Sarcosine oxidase（SOx）With horseradish peroxidase（HRP）Assembled respectively with DNA：By SOx, HRP respectively with mistake Bridging agent 3- (2- pyridines dimercapto) propionic acid N-hydroxy-succinamide ester of amount（SPDP）After 25 DEG C of mixing is incubated 2 hr, ultrafiltration Wash away excessive SPDP；The DNA 1 with sulfydryl modification, DNA 2 are uniform with certain proportion respectively for two kinds of enzymes of subsequent SPDP modifications 25 DEG C of overnight incubations, wash away excessive DNA using ultrafiltration afterwards, obtain enzyme-DNA compound SOx-DNA complexⅠs and HRP-DNA Complexⅱ；
    （2）The synthesis of tubulose DNA paper foldings：Equal proportion is well mixed to replace chain DNA and original DNA staples chain, described DNA foldings Paper is tubulose DNA paper foldings, in 1 × TAE-Mg2+In solution, staple chain is well mixed with long-chain single stranded DNA with certain proportion After annealing forms DNA origami structures；Excessive staple chain is washed away using ultrafiltration, DNA is respectively obtained according to chain difference is replaced Origami I and DNA origami II, wherein described long-chain single stranded DNA is the conventional long-chain in this area；
    （3）Enzyme-DNA compounds assemble with two kinds of DNA paper foldings respectively：SOx-DNA complexⅠs (or HRP-DNA complexⅱs) with DNA origami I or DNA origami II are well mixed with certain proportion, in 45 DEG C of annealing assemblings；Pass through Ago-Gel Electrophoretic separation removes the enzyme to dissociate in annealed product and paper folding, obtains containing SOx-origami compounds III and HRP-origami The band of compound IV, removing glue, recovery SOx-origami compounds III and HRP-origami compounds are purified using Filter column Ⅳ；
    （4）The assembling of SOx-origami compounds III and HRP-origami compounds IV：By the He of SOx-origami compounds III HRP-origami compounds IV are well mixed with certain proportion, and in 37 DEG C of annealing assemblings, it is compound to obtain SOx-origami-HRP Thing V；
    （5）Methyl amimoacetic acid detects：The reaction mother liquor of methyl amimoacetic acid and HRP catalyzed coloration substrates is configured in advance, by finite concentration during detection Methyl amimoacetic acid and chromogenic substrate are well mixed, and are added the SOx-origami-HRP compounds V of certain content, are selected according to chromogenic substrate Suitable instrument is selected to be measured.
  2. 2. the method for prostate cancer target methyl amimoacetic acid is detected according to claim 1, it is characterised in that in described buffer solution, Enzyme modification DNA and detection when preferably buffer it is molten for pH=7.4,8 mM Na2HPO4、2 mM KH2PO4、136 mM Na Cl、 2.6 mM K Cl, 1 × PBS；The buffer solution of DNA paper foldings synthesis and agarose gel electrophoresis is pH=8.0,40 mM Tris- acetic acid, 1 mM EDTA, 12.5 mM MgCl2 1×TAE-Mg2+Solution.
  3. 3. the method for prostate cancer target methyl amimoacetic acid is detected according to claim 1, it is characterised in that described enzyme and DNA's Preferable ratio is assembled as 1:10；Described tubulose DNA paper foldings, preferable a width of 6 nm of tubulose DNA paper foldings, the length of side 412 nm；Described long-chain single stranded DNA is preferably M13mp18 single stranded DNAs.
  4. 4. according to the method for the detection prostate cancer target methyl amimoacetic acid of claim 1 or 3, it is characterised in that step（2）In Paper folding assembling mode comprises the following steps：Single-stranded M13mp18 and the staple chain DNA are mixed in 1 × TAE-Mg2+Buffer molten In liquid, 20 DEG C are annealed to from 90 DEG C in PCR instrument, annealing rate is 0.1 DEG C/10s.
  5. 5. the method for prostate cancer target methyl amimoacetic acid is detected according to claim 1, it is characterised in that wrapped in described DNA Seq. No.1 to 22 DNA is included, wherein, with the 3 ' ends that the complementary DNA 3 of the Sequences of DNA 1 is the Column 1 of Helix 4 Hold the sequence extended；With the sequence of the complementary DNA 4 of the Sequences of the DNA 23 ' terminal extensions for being the Column 1 of Helix 4； The sequence DNA 5-13 replaced in addition is respectively the Column 5/14/23/32/38/47/56/65/74 of Helix 13 ' ends The sequence of extension；The sequence DNA 14-22 of replacement is respectively the Column 6/15/24/33/39/48/57/66/75 of Helix 6 3 ' terminal extensions sequence；The sequence of wherein DNA 5-13 3 ' end sequences and DNA 14-22 3 ' terminal extensions is complementary；
    Described SOx-DNA complexⅠs and HRP-DNA complexⅱ assembles with DNA origami I and DNA origami II Mode preferably 20 DEG C are annealed to from 45 DEG C in PCR instrument, annealing rate is 0.1 DEG C/15 s.
  6. 6. according to the method for the detection prostate cancer target methyl amimoacetic acid of claim 1 or 5, it is characterised in that described SOx- The ratio of DNA complexⅠs and HRP-DNA complexⅱ and DNA origami I and DNA origami II assembling preferably compares Example is SOx-DNA complexⅠs or HRP-DNA complexⅱs：DNA origami I or DNA origami II=10:1.
  7. 7. the method for prostate cancer target methyl amimoacetic acid is detected according to claim 1, it is characterised in that step（3）In, it is described SOx-origami compounds III and the way of purification of HRP-origami compounds IV be preferably agarose gel electrophoresis, fine jade Lipolysaccharide concentration is 0.5 %, and deposition condition is the min of 80 V voltage ice baths electrophoresis 80；To the He of SOx-origami compounds III The way of recycling of the band of HRP-origami compounds IV is preferably pure using Freeze N Squeeze spin columns Change.
  8. 8. the method for prostate cancer target methyl amimoacetic acid is detected according to claim 1, it is characterised in that step（4）In, it is described SOx-origami compounds III and HRP-origami IV the preferable ratio of assembling ratio be 1:1；Assembling mode is preferable Using being annealed to 10 DEG C from 37 DEG C in PCR instrument, annealing rate is 0.1 DEG C/15 s.
  9. 9. the method for prostate cancer target methyl amimoacetic acid is detected according to claim 1, it is characterised in that step（5）In, it is described HRP catalyzed coloration substrates be preferably 2,2- connection nitrogen-two (3- ethyls-benzothiazole -6- sulfonic acid) di-ammonium salts（ABTS）, 3, 3', 5,5'- tetramethyl benzidine（TMB）, 10- acetyl group -3,7- dihydroxyphenazines（Amplex Red）, will be certain dense during detection Degree methyl amimoacetic acid and chromogenic substrate are well mixed.
  10. 10. according to the method for the detection prostate cancer target methyl amimoacetic acid of claim 1 or 9, it is characterised in that step（5）In, The preferred concentration of the concentration of SOx-origami-HRP compounds V is 1 nM during detection.
CN201711085501.4A 2017-11-07 2017-11-07 The method for detecting prostate cancer target methyl amimoacetic acid Pending CN107760759A (en)

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