CN101526467B - Optical biological sensing technology based on interaction between terminal-protected analyzed detected small molecules of single-chain exonuclease I and combined protein - Google Patents
Optical biological sensing technology based on interaction between terminal-protected analyzed detected small molecules of single-chain exonuclease I and combined protein Download PDFInfo
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- CN101526467B CN101526467B CN2009100431321A CN200910043132A CN101526467B CN 101526467 B CN101526467 B CN 101526467B CN 2009100431321 A CN2009100431321 A CN 2009100431321A CN 200910043132 A CN200910043132 A CN 200910043132A CN 101526467 B CN101526467 B CN 101526467B
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Abstract
The invention discloses an optical biological sensing technology based on the interaction between terminal-protected analyzed detected small molecules of single-chain exonuclease I and a combined protein. The technology comprises the following steps: (1), the interaction between an oligonucleotide DNA single chain modified by terminal organic small molecules and containing a hemachrome aptamer sequence and the protein and the protective analysis of the exonuclease I; and (2) the color development and the chemiluminescent detection of the terminal-protected oligonucleotide DNA single chain containing the hemachrome aptamer sequence. The method has high sensitivity, simple and convenient operation and strong specificity, can be jointly used with an enzyme mark instrument generally used in the prior biological detection and can be used for visual detection so as to be a general technology used for signal transduction and molecular regulation and control mechanism research in biomedicine,the safety detection of food and agricultural products, environmental poison detection and drug screening.
Description
Technical field
The invention belongs to a kind of optical bio sensing technology that detects organic molecule and binding protein interactions, comprise the interaction and the exonuclease I protection analysis of the oligonucleotide DNA strand and the albumen of terminal organic molecule modification.
Background technology
Organic molecule such as organic molecule and binding protein interactions, medicine and chemical toxicant and the protein-bonded rapid screening of micromolecule are extremely important for fields such as clinical diagnosis, medical research, food and public safety, drug screening, environmental monitorings with detecting.The organic molecule commonly used at present and the detection technique of binding protein interactions mainly contain surface plasma resonance, the complementary segment immunoassay of enzyme and fluorescence anisotropy analysis etc.Not high, the poor reliability of these detection technique sensitivity, and need accurate instrument, can not satisfy the demand of accurate fast detecting.
Summary of the invention
The technical problem to be solved in the present invention is; deficiency at the prior art existence; a kind of optical bio sensing technology based on single-chain nucleic acid excision enzyme I terminal protection analyzing and testing micromolecule and binding protein interactions is proposed; it does not have dependence to dna sequence dna; only require that this sequence comprises the protoheme aptamer; and the catalytic activity that this nucleotide sequence has the protoheme aptamer gets final product; thereby can utilize the superoxide enzymatic activity of protoheme aptamer, carry out the detection by quantitative that DNA excision enzyme I handles the oligonucleotide DNA strand of back protection by catalysis chromogenic reaction and chemiluminescence response.
Technical scheme of the present invention is that described optical bio sensing technology based on single-chain nucleic acid excision enzyme I terminal protection analyzing and testing micromolecule and binding protein interactions comprises:
(1) interaction of the oligonucleotide DNA strand that contains the fit sequence of protoheme of terminal organic molecule modification and albumen and exonuclease I protection are analyzed;
(2) colour developing and the chemiluminescence detection of the oligonucleotide DNA strand that contains the fit sequence of protoheme of terminal protection.
Below the present invention made further specify.
The oligonucleotide DNA strand that contains the fit sequence of protoheme that described terminal organic molecule is modified and the interaction of albumen and exonuclease I protection are analyzed and are:
For micromolecule interaction protein-bonded, detect step and be: get described organic molecule and modify the storing solution of the oligonucleotide DNA strand that contains the fit sequence of protoheme and place micro tube with it; In micro tube, add again and comprise the protein-bonded sample solution of micromolecule; Add exonuclease I reaction then, get the product of terminal protective reaction;
For the detection of organic molecule, adopt competitive reaction to detect step: to get described organic molecule and modify the storing solution of the oligonucleotide DNA strand that contains the fit sequence of protoheme and place micro tube; Add again and comprise micromolecular sample solution to be measured, mix the back and add micromolecule in conjunction with albumen or monoclonal anti liquid solution; Add exonuclease I again, get the product of terminal protective reaction.
The colour developing and the chemiluminescence detection of the oligonucleotide DNA strand that contains the fit sequence of protoheme of described terminal protection can adopt one of following method:
(1) absorption spectrophotometry detects: add protoheme and 2 in the product of described terminal protective reaction, two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts of 2 '-Lian nitrogen, add hydrogen peroxide again, survey the absorbance of its random wave strong point in the 300-500nm scope with ultraviolet spectrophotometer;
(2) chemoluminescence method detects: in the product of described terminal protective reaction, add protoheme and luminol, and hydrogen peroxide again, the random wave strong point writes down its chemiluminescence intensity in the 300-500nm scope.
Among the present invention, the modification of the terminal organic molecule of the described oligonucleotide DNA strand that comprises the protoheme aptamer realizes by existing cross-linking reaction technology.For example, for the organic molecule that has carboxyl, can adopt 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide and succinimide cross-linking reaction technology and 3 ' end NH
2The oligonucleotide DNA strand that comprises the protoheme aptamer of mark carries out crosslinked; For the amino organic molecule of band, the cross-linking reaction technology that can adopt 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester is carried out crosslinked with the oligonucleotide DNA strand that comprises the protoheme aptamer that is marked with sulfydryl with 3 ' end.The cross-linking reaction product can be used the dialysis purifying.
Further, among the present invention, the oligonucleotide DNA strand that described terminal organic molecule is modified and the interaction of albumen and exonuclease I protection analytical technology are:
A. for the micromolecule step that protein-bonded interactional detection and exonuclease I protection is analyzed with it be: get that the described organic molecule of 5 μ L is modified and the storing solution that comprises the oligonucleotide DNA strand of protoheme aptamer places micro tube, add 12.5 μ L, 2 * HEPES buffer solution, this buffer solution is 25mM HEPES, 50mM NaCl, 50mM KCl, pH 7.0; Add 5 μ L again and comprise the protein-bonded sample solution of micromolecule, 37 ℃ of isothermal reactions 0.5 hour, add 0.5 μ l exonuclease I then, place 37 ℃ of reactions after 5 minutes, be warming up to 80 ℃ of reactions and carried out hot deactivation in 20 minutes, get the solution a after exonuclease is handled to stop enzyme reaction;
B. analyzing the competitive reaction of adopting for the detection of organic molecule and exonuclease I protection detects step and is: get that the described organic molecule of 5 μ L is modified and the storing solution that comprises the oligonucleotide DNA strand of protoheme aptamer places micro tube, add 12.5 μ L, 2 * HEPES buffer solution, this buffer solution is 25mM HEPES, 50mM NaCl, 50mM KCl, pH 7.0; Add 5 μ L again and comprise micromolecular sample solution to be measured, mixing the back adds certain density micromolecular in conjunction with albumen or (mouse source) monoclonal antibody 3 μ L, micromolecular in conjunction with for the organic molecule modified oligonucleotide dna single chain concentration that adds 5 times of albumen or the whole N of (mouse source) monoclonal antibody; 37 ℃ of isothermal reactions 0.5 hour; Add 0.5 μ L exonuclease I then, place 37 ℃ of reactions after 5 minutes, be warming up to 80 ℃ of reactions 20 minutes, carry out hot deactivation, get the solution b after exonuclease is handled to stop enzyme reaction.
Notice that above reaction conditions is an optimal conditions, described liquor capacity all can change at double and not change optimal result.Change ratio or reagent addition sequence can make the protection efficient of organic molecule modified oligonucleotide dna single chain change in 3%～100%.
Among the present invention, the colour developing and the chemiluminescence detection of the oligonucleotide DNA strand of described terminal protection can adopt one of following method:
(1) absorption spectrophotometry detects: the protoheme 2 μ L that add 500nM among the solution b after solution a after described exonuclease is handled or exonuclease are handled, room temperature reaction 10 minutes, add 1 μ L 50mM 2 subsequently, two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts (ABTS) of 2 '-Lian nitrogen, add 2 * HEPES buffer solution, 30 μ L and aqua sterilisa 30 μ L again, and add 1% hydrogen peroxide (H
2O
2) 2 μ L, survey its absorbance with ultraviolet spectrophotometer at 414nm; Described buffer solution is 25mM HEPES, 200mM NaCl, and 20mM KCl, pH 8.0; Notice that the above reaction conditions is an optimal conditions, described liquor capacity all can change at double and not change detection sensitivity.Change ratio or reagent addition sequence can make sensitivity change variation in 0.1%～100% relatively.
(2) chemoluminescence method detects: the protoheme 2 μ L that add 500nM among the solution b after solution a after described exonuclease is handled or exonuclease are handled, room temperature reaction 10 minutes, adding 5 μ L concentration subsequently is the luminol of 50 μ M, add 2 * HEPES buffer solution, 12.5 μ L and aqua sterilisa 5 μ L again, and add 1% hydrogen peroxide (H
2O
2) 2 μ L, in 425nm place its chemiluminescence intensity value of record and definite maximum luminescence peak; Described buffer solution is 25mMHEPES, 200mM NaCl, and 20mM KCl, pH 8.0.Notice that the above reaction conditions is an optimal conditions, described liquor capacity all can change at double and not change detection sensitivity.Change ratio or reagent addition sequence can make sensitivity change variation in 1%～100% relatively.
The optical bio sensing technology that the present invention sets up based on exonuclease I end points protection analyzing and testing organic molecule and binding protein interactions, utilize the superoxide enzymatic activity of protoheme aptamer, carry out the detection by quantitative that DNA excision enzyme I handles the oligonucleotide DNA strand of back protection by catalysis chromogenic reaction and chemiluminescence response; This technology also can be directly used in the protein-bonded detection of micromolecule, also can be by the competition of oligonucleotide DNA strand and antibodies indirect detection organic molecule of micromolecule in the sample solution and micromolecule mark.It is highly sensitive, operates fast and conveniently, and being expected provides a current techique platform for clinical diagnosis, medicament research and development, medicine toxicological analysis, environmental monitoring etc.
Embodiment:
Embodiment 1: based on the terminal protection of exonuclease I analyzing and testing Ractopamine
1) 3 ' end NH
2The oligonucleotide DNA strand of mark and Ractopamine crosslinked:
The hydrochloric acid Ractopamine and the 12mg succinic anhydride that take by weighing 34mg are dissolved in the 2ml pyridine, react 24h under the room temperature, follow the tracks of reaction (developping agent: 90% methylene chloride, 10% the methyl alcohol that contains 10% ammoniacal liquor) with thin-layer chromatography, and product is through the Rotary Evaporators evaporate to dryness.Take by weighing product 10.6mg and be dissolved in 2.5mL phosphate buffered solution (0.1M NaH2PO4, pH 7.4), add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) of 1mg again, at room temperature stir 15min, add 2.8mg succinimide (NHS) again, at room temperature reaction 30min.Getting biotin solution after the 260 μ l activation joins in the solution that 260 μ L concentration are 3 ' the end NH2 mark of the 1 μ M oligonucleotide DNA strand (sequence is GGGTAGGGCGGGTTGGGTTTTTTTTTTTTTTTTTTTTTT) that comprises the protoheme aptamer, transferring its pH with the NaOH of 1M is 7.4, room temperature reaction 2h under gentle agitation.The reaction back adds 3.6mg oxammonium hydrochloride cessation reaction in mixed liquor.
The cross-linking reaction product is moved on in the dialysis tubing of 1mL, the molecular cut off of dialysis tubing is 6000D.Dialysis tubing is put in the 500ml phosphate buffered solution (0.1M NaH2PO4, pH 7.4) at 4 ℃ of dialysis 12h, changed a dislysate, dialyse with ultrapure water for the last time every 3h.Oligonucleotide DNA strand after the dialysis is in charge of in the refrigerator that is stored in-20 ℃ standby.The oligonucleotide DNA strand of-20 ℃ of above-mentioned preservations is with 10 times of aqua sterilisa dilutions and be stored in 4 ℃ of refrigerators standby as secondary storing solution.
2) detection of Ractopamine:
The secondary storing solution of oligonucleotide DNA strand of getting the above-mentioned Ractopamine mark of 5 μ l is in micro tube, the concentration that makes final DNA oligonucleotide chain is 400nM, add 2 * HEPES buffer solution (25mM HEPES, 50mM NaCl, 50mM KCl, pH 7.0) 12.5 μ L, add 5 μ L Ractopamine sample solution to be detected again, mixing back adding 2.5 μ L concentration is the Ractopamine mouse resource monoclonal antibody of 2000nM, room temperature reaction 45min, the reaction back adds 1 μ L exonuclease I (NEB company product) after 37 ℃ of enzymes are cut 5min, is warming up to 80 ℃ of reaction 20min and carries out hot deactivation to stop enzyme reaction.
The protoheme 2 μ L that add 500nM in the solution after above-mentioned exonuclease is handled, room temperature reaction 10min, add 1 μ L 50mM 2 subsequently, two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts (ABTS) of 2 '-Lian nitrogen, add 2 * HEPES buffer solution (25mM HEPES, 200mM NaCl, 20mM KCl again, pH 8.0) 30 μ L and aqua sterilisa 30 μ L, add 1% hydrogen peroxide (H rapidly
2O
2) 2 μ L, survey its absorbance with ultraviolet spectrophotometer at 414nm.
By above-mentioned 1), 2) (concentration is from 10 to the Ractopamine standard solution of 6 preparations for step
-10M to 10
-5M is every one of an order of magnitude) detect, write down the absorbance of each standard solution sample, with absorbance Ractopamine concentration mapping in the standard solution is obtained typical curve.
Embodiment 2: based on exonuclease end points protection analyzing and testing FABP
1) 3 ' end NH
2The oligonucleotide DNA strand of mark and folic acid crosslinked:
The folic acid that takes by weighing 10mg is dissolved in 2.5mL phosphate buffered solution (0.1M NaH
2PO4, pH 7.4), add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) of 1mg again, at room temperature stir 15min, add 2.8mg succinimide (NHS) again, at room temperature reaction 30min.The folic acid solution of getting after 260 μ l activate joins 3 ' the end NH that 260 μ l concentration are 10 μ M
2In the solution of the oligonucleotide DNA strand of mark (sequence is GGGTAGGGCGGGTTGGGTTTTTTTTTTTTTTTTTTTTTT), transferring its pH with the NaOH of 1M is 7.4, room temperature reaction 2h under gentle agitation.The reaction back adds 3.6mg oxammonium hydrochloride cessation reaction in mixed liquor.
The cross-linking reaction product is moved on in the dialysis tubing of 1mL, the molecular cut off of dialysis tubing is 6000D.Dialysis tubing is put into 500ml phosphate buffered solution (0.1M NaH
2PO4, pH 7.4) at 4 ℃ of dialysis 12h, change a dislysate every 3h, dialyse with ultrapure water for the last time.Oligonucleotide DNA strand after the dialysis is in charge of in the refrigerator that is stored in-20 ℃ standby.The oligonucleotide DNA strand of-20 ℃ of above-mentioned preservations is with 10 times of aqua sterilisa dilutions and be stored in 4 ℃ of refrigerators standby as secondary storing solution.More than all reactions all under the condition of shading, carry out.
2) detection of FABP:
The secondary storing solution of oligonucleotide DNA strand of getting the above-mentioned folic acid mark of 7.5 μ l is in micro tube, the concentration that makes final DNA oligonucleotide chain is 100nM, add 2 * HEPES buffer solution (25mM HEPES, 50mM NaCl, 50mM KCl, pH 7.0) 12.5 μ L, add 5 μ L FABP sample solution to be detected again, room temperature reaction 45min, the reaction back adds 0.5 μ L exonuclease I (NEB company product) after 37 ℃ of enzymes are cut 5min, is warming up to 80 ℃ of reaction 20min and carries out hot deactivation to stop enzyme reaction.
The protoheme 2 μ L that add 500nM in the solution after above-mentioned exonuclease is handled, room temperature reaction 10min, adding 5 μ L concentration subsequently is the luminol of 50 μ M, add 2 * HEPES buffer solution (25mM HEPES again, 200mMNaCl, 20mM KCl, pH 8.0) 12.5 μ L and aqua sterilisa 5 μ L, add 1% hydrogen peroxide (H rapidly
2O
2) 2 μ L, in 425nm place its chemiluminescence intensity value of record and definite maximum luminescence peak.
By above-mentioned 1), 2) (concentration is from 10 to the FABP standard solution of 7 preparations for step
-15M to 10
-9M is every one of an order of magnitude) detect, write down the chemiluminescence peak value of each standard solution sample, with the chemiluminescence peak value FABP concentration mapping in the standard solution is obtained typical curve.
Claims (1)
1. optical bio method for sensing based on single-chain nucleic acid excision enzyme I terminal protection analyzing and testing micromolecule and binding protein interactions comprises:
(1) interaction of the oligonucleotide DNA strand that contains the fit sequence of protoheme of terminal organic molecule modification and albumen and exonuclease I protection oligonucleotide DNA strand are analyzed the oligonucleotide DNA strand of being protected then;
For micromolecule interaction protein-bonded, detect step and be: get described organic molecule and modify the storing solution of the oligonucleotide DNA strand that contains the fit sequence of protoheme and place micro tube with it; In micro tube, add again and comprise micromolecular protein-bonded sample solution; Add exonuclease I reaction then, get the product of terminal protective reaction;
For micromolecular detection to be measured, adopt competitive reaction to detect step: to get described organic molecule and modify the storing solution of the oligonucleotide DNA strand that contains the fit sequence of protoheme and place micro tube; Add again and comprise micromolecular sample solution to be measured, mix the back and add micromolecular in conjunction with albumen or monoclonal anti liquid solution; Add exonuclease I again, get the product of terminal protective reaction;
(2) based on micromolecular in conjunction with the end protection of albumen to the oligonucleotide DNA strand that is marked with the fit sequence of micromolecular protoheme, with colour developing and chemiluminescence detecting method the oligonucleotide DNA strand of being protected that contains the fit sequence of protoheme is detected; One of following method is adopted in described detection:
A. absorption spectrophotometry detects: add protoheme and 2 in the product of described terminal protective reaction, two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts of 2 '-Lian nitrogen, add hydrogen peroxide again, survey the absorbance of its random wave strong point in the 300-500nm scope with ultraviolet spectrophotometer;
B. chemoluminescence method detects: add protoheme and luminol in the product of described terminal protective reaction, add hydrogen peroxide again, the random wave strong point writes down its chemiluminescence intensity in the 300-500nm scope, and determines maximum luminescence peak.
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