CN104178568A - Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe - Google Patents
Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe Download PDFInfo
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- CN104178568A CN104178568A CN201410360025.2A CN201410360025A CN104178568A CN 104178568 A CN104178568 A CN 104178568A CN 201410360025 A CN201410360025 A CN 201410360025A CN 104178568 A CN104178568 A CN 104178568A
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Abstract
The invention discloses a method for detecting a target substance in a to-be-detected sample based on fluorescent sensing analysis of an aptamer probe. The method comprises the following steps: (1) jointly incubating functional magnetic beads and the to-be-detected sample together, wherein the functional magnetic beads are obtained by hybridizing magnetic beads (a) of which the surfaces are modified with specifically recognized target substance and a probe (b) linked with streptavidin and a marker; (2), after the step (1) is completed, carrying out magnetic separation and collecting a supernatant; and (3) under the excitation effect of an excitation wavelength corresponding to the marker, detecting the supernatant obtained in the step (2) by virtue of a solid-phase chip of which the surface is modified with a dethiobiotin or a biotin and determining whether the target substance is contained in the to-be-detected sample according to the values of signals. The method disclosed by the invention is low in cost and simple and rapid and has the advantages of wide aptamer target molecules, good specificity and high sensitivity and the method can be stably used over 300 times.
Description
Technical field
The present invention relates to a kind of method that detects the target substance in sample to be tested based on aptamer fluorescence probe sensing assays.
Background technology
The sensing analytical method that the aptamer of take is bio-identification material has obtained develop rapidly in nearest two more than ten years.Aptamer be one section can specific recognition target contaminant RNA or single stranded DNA fragment.As far back as eighties of last century the nineties, Nature and Science periodical almost simultaneously reported first aptamer material and triage techniques thereof.Through the development of twenties years, the aptamer material of having reported at present hundreds of material in can testing environment.
The sensing analytical method that the aptamer of take is biological identification element comprises can be divided into homogeneous phase and two kinds of methods of heterogeneous (or out-phase) aptamers sensing assays.At present, the aptamer sensing technology of homogeneous phase method remains main flow.Such as, LuYi seminar of University of Illinois at Urbana-Champaign university utilizes on aptamer probe fixedly fluorophor and quencher group, DNA conformational change based on producing after target substance identification realizes the enhancing of fluorescent signal or weakens, thereby sets up the quantitative relationship of target concentration and fluorescent signal.Homogeneous reaction speed is fast, and testing process is simple, yet reagent consumption is large, testing cost is high.The fixedly ssDNA probe of conventionally take solid phase method, as main, detects the difference realization of hybridization chain and target affinity based on DNA probe.For realizing repeatedly, use, need to be by DNA chain or target wash-out after hybridization.The elution process of having reported comprises: pickling/alkali cleaning, heating, high eluting salt, EDTA chelating, tensio-active agent wash-out etc.Shortcoming is that to stablize reusable number of times very limited, conventionally between 5-15 time.Trace it to its cause is, a little less than three grades of conformations of DNA are highly brittle, under various elution requirements, to be difficult to return to original state.Low so stable development and the scope of application that number of times has also greatly limited solid phase DNA technique of reusing.
Summary of the invention
The object of this invention is to provide a kind of method that detects the target substance in sample to be tested based on aptamer fluorescence probe sensing assays.
The invention provides a kind of method that whether contains target substance in sample to be tested that detects, comprise the steps:
(1) function magnetic bead and described sample to be tested are hatched altogether; Described function magnetic bead is hybridized and is obtained by (a) with (b); Described (a) has the magnetic bead of the aptamer of specific recognition target substance for finishing; Described (b) is for being connected with the probe of Streptavidin and marker; Described probe is the single stranded nucleic acid molecule complementary with described aptamer part;
(2) after completing steps (1), carry out magnetic separation, collect supernatant liquor;
(3), under exciting with the corresponding excitation wavelength of marker, the supernatant liquor that adopts finishing to have the solid phase chip detecting step (2) of desthiobiotin or vitamin H to obtain, judges in sample to be tested, whether to contain target substance according to signal value.
Described finishing has the preparation method of the solid phase chip of desthiobiotin to comprise the steps:
(I) gets solid phase chip, makes its surface hydroxylation;
After (II) completing steps (I), get solid phase chip, make its finishing APTS;
After (IV) completing steps (II), get solid phase chip, make its finishing desthiobiotin.
The method of " getting solid phase chip, making its surface hydroxylation " is specific as follows: 1. solid phase chip is immersed to piraha solution (98% dense H
2sO
4: H
2o
2=3:1, volume ratio) and 120 ℃ of reaction 1h; 2. completing steps 1. after, take out solid phase chip, with ultrapure water, clean until pH value is neutrality, at room temperature with nitrogen, dry up, be then placed in 70 ℃ of vacuum drying ovens and preserve 1h.
The method of " getting solid phase chip, making its finishing APTS " is specific as follows: 1. get solid phase chip, be placed in APTS solution and react 1h; 2. completing steps 1. after, take out solid phase chip, with dry toluene, rinse, at room temperature with nitrogen, dry up, be then placed in 200 ℃ of baking 1h.APTS solution: solute is APTS, solvent is dry toluene, the concentration of solute is 2% (volume ratio).
The method of " getting solid phase chip, making its finishing desthiobiotin " is specific as follows: 1. get solid phase chip, be placed in glutaraldehyde solution room temperature reaction 1h, then use alcohol flushing, be then placed in ethylenediamine solution room temperature reaction 1.5h, be then placed in NaBH
4room temperature reaction 15min in solution; 2. get 25mg desthiobiotin, 50mg EDC and 50mg NHS, with 1000 μ l DMF, dissolve, room temperature reaction 3h, then adds the NaHCO of 200 μ l pH8.7,1M
3-Na
2cO
3damping fluid also mixes, and obtains mixed solution; 3. completing steps 1. after, take out solid phase chip, add in the mixed solution that 2. step obtain room temperature reaction 12 hours; 4. completing steps 3. after, take out solid phase chip, with alcohol flushing, be then placed in the PBS damping fluid containing 2mg/mL BSA, room temperature reaction 1h.Glutaraldehyde solution: solute is glutaraldehyde, solvent is ethanol, the concentration of volume percent of solute is 2.5%.Ethylenediamine solution: solute is quadrol, solvent is ethanol, the concentration of volume percent of solute is 10%.NaBH
4solution: solute is NaBH
4, solvent is ethanol, the concentration of solute is 10mg/ml.
The preparation method of described " being connected with the probe of Streptavidin and marker " is specific as follows: 1. synthesising probing needle, at 5 ' end, carry out sulfydryl modification, and at 3 ' end, carry out marker modification, called after SH-DNA-marker; 2. in 10 μ l 1mM SH-DNA-markers, add 0.67 μ L TCEP solution, lucifuge room temperature reaction 1h, to activate sulfydryl; 3. get Amicon-3K, the whole reaction system that adds completing steps 2. to obtain afterwards, then add 200 μ L Buffer A-1, the then centrifugal 10min of 12000rpm, with buffer A-2 washing Amicon-3K, collect the component (solution form) that molecular weight is greater than 3K; 4. get the 133 μ L 20mg/mL Streptavidin aqueous solution, add 0.33mg Sulfo-SMCC, whirlpool concussion 5min, room temperature reaction 1h then, the centrifugal 5min of 12000rpm, gets supernatant liquor; 5. get Amicon-10K, the supernatant liquor that adds step 4. to obtain, then add 200 μ L Buffer A-1, the centrifugal 10min of 12000rpm, with buffer A-2 washing Amicon-10K, collects the component (solution form) that molecular weight is greater than 10K; 6. the solution 5. step being obtained adds in the solution that 3. step obtain, lucifuge room temperature reaction 48h; 7. get Amicon-10K, the whole reaction system that adds completing steps 6. to obtain afterwards, add again 200 μ L Buffer A-1, the centrifugal 10min of 12000rpm, with buffer A-2 washing Amicon-10K, collection molecular weight is greater than the component (solution form) of 10K, is and contains the probe solution that is connected with Streptavidin and marker.TCEP solution: solute is TCEP, solvent ultrapure water, the concentration of solute is 30mM.Buffer A-1: containing the aqueous solution of 0.1M NaCl and 0.1M sodium phosphate, pH7.3.Buffer A-2: containing the aqueous solution of 0.1M NaCl, 0.1M sodium phosphate and 0.05% (volume ratio) Tween 20, pH7.3.
The preparation method of described " finishing has the magnetic bead of the aptamer of specific recognition target substance " comprises the steps: 1. 38.4mg EDC to be dissolved in the Methylimidazole damping fluid of 2mL pH 7.0,0.1M, then add aptamer described in 0.65nmol, fully mix, obtain mixed solution; 2. the mixed solution 1. step being obtained adds in about 20mg magnetic bead, room temperature shakes up reaction 24 hours, then magnetic is separated and collect magnetic bead, with 2 milliliters of prehybridization damping fluids, the unreacted carboxyl of magnetic bead surfaces is sealed, and obtains the magnetic bead that finishing has the aptamer of specific recognition target substance.Prehybridization damping fluid: solvent is the Tris damping fluid of pH7.4,0.1M, containing 0.005M EDTA, 0.5% (volume ratio) N-Sarkosyl L and 1g/100mL BSA.
The preparation method of described function magnetic bead specifically comprises the steps: 1. to get the magnetic bead that about 20mg finishing has the aptamer of specific recognition target substance, with 2mL hybridization buffer, suspends, and in 60 ℃ of water-baths, temperature is bathed 1h; 2. get 10 μ L and contain the probe solution that is connected with Streptavidin and marker, be dissolved in 2mL hybridization buffer, in 60 ℃ of water-baths, temperature is bathed 1h; 3. the solution that 2. bead suspension 1. step being obtained and step obtain mixes, and room temperature shakes up reaction 2.5h, obtains function bead suspension.Hybridization buffer: containing 10mM Tris, 120mM NaCl, 5mM KCl, 20mM CaCl
2the aqueous solution, pH 8.5.
Described marker specifically can be fluorescent marker, more specifically can be Cy5.5.The corresponding excitation wavelength of described and marker specifically can be 650nm.
Described step (3) is specifically carried out in biosensor of full fiber optic evanescent wave.The working specification of biosensor of full fiber optic evanescent wave is as follows: open laser apparatus, excitation wavelength is 650nm; With PBS damping fluid, rinse finishing and have the solid phase chip of desthiobiotin or vitamin H until baseline is steady; After baseline is steady, get solution to be measured, open peristaltic pump sample introduction 25s, stop afterwards peristaltic pump, make solution to be measured in reaction tank, react 120s; Again open peristaltic pump, pass into elutriant (the 0.5g/100mL SDS aqueous solution is adjusted pH to 1.9) 90 seconds; Finally pass into PBS damping fluid, steady to baseline.
Described magnetic bead specifically can be carboxyl magnetic bead.
Described solid phase chip specifically can be optical fiber.
Described target substance is Ochratoxin A.
Described aptamer is specifically as shown in the sequence 1 of sequence table.Described probe is specifically as shown in the sequence 2 of sequence table.
The present invention also protects a kind of test kit that whether contains target substance in sample to be tested that detects, and comprises that function magnetic bead and finishing have the solid phase chip of desthiobiotin or vitamin H; Described function magnetic bead is hybridized and is obtained by (a) with (b); Described (a) has the magnetic bead of the aptamer of specific recognition target substance for finishing; Described (b) is for being connected with the probe of Streptavidin and marker; Described probe is the single stranded nucleic acid molecule complementary with described aptamer part.
The present invention also protects a kind of test kit that whether contains target substance in sample to be tested that detects, and comprises " solid phase chip that finishing has desthiobiotin or vitamin H ", " finishing has the magnetic bead of the aptamer of specific recognition target substance " and " being connected with the probe of Streptavidin and marker "; Described probe is the single stranded nucleic acid molecule complementary with described aptamer part.
Described finishing has the preparation method of the solid phase chip of desthiobiotin to comprise the steps:
(I) gets solid phase chip, makes its surface hydroxylation;
After (II) completing steps (I), get solid phase chip, make its finishing APTS;
After (IV) completing steps (II), get solid phase chip, make its finishing desthiobiotin.
The method of " getting solid phase chip, making its surface hydroxylation " is specific as follows: 1. solid phase chip is immersed to piraha solution (98% dense H
2sO
4: H
2o
2=3:1, volume ratio) and 120 ℃ of reaction 1h; 2. completing steps 1. after, take out solid phase chip, with ultrapure water, clean until pH value is neutrality, at room temperature with nitrogen, dry up, be then placed in 70 ℃ of vacuum drying ovens and preserve 1h.
The method of " getting solid phase chip, making its finishing APTS " is specific as follows: 1. get solid phase chip, be placed in APTS solution and react 1h; 2. completing steps 1. after, take out solid phase chip, with dry toluene, rinse, at room temperature with nitrogen, dry up, be then placed in 200 ℃ of baking 1h.APTS solution: solute is APTS, solvent is dry toluene, the concentration of solute is 2% (volume ratio).
The method of " getting solid phase chip, making its finishing desthiobiotin " is specific as follows: 1. get solid phase chip, be placed in glutaraldehyde solution room temperature reaction 1h, then use alcohol flushing, be then placed in ethylenediamine solution room temperature reaction 1.5h, be then placed in NaBH
4room temperature reaction 15min in solution; 2. get 25mg desthiobiotin, 50mg EDC and 50mg NHS, with 1000 μ l DMF, dissolve, room temperature reaction 3h, then adds the NaHCO of 200 μ l pH8.7,1M
3-Na
2cO
3damping fluid also mixes, and obtains mixed solution; 3. completing steps 1. after, take out solid phase chip, add in the mixed solution that 2. step obtain room temperature reaction 12 hours; 4. completing steps 3. after, take out solid phase chip, with alcohol flushing, be then placed in the PBS damping fluid containing 2mg/mL BSA, room temperature reaction 1h.Glutaraldehyde solution: solute is glutaraldehyde, solvent is ethanol, the concentration of volume percent of solute is 2.5%.Ethylenediamine solution: solute is quadrol, solvent is ethanol, the concentration of volume percent of solute is 10%.NaBH
4solution: solute is NaBH
4, solvent is ethanol, the concentration of solute is 10mg/ml.
The preparation method of described " being connected with the probe of Streptavidin and marker " is specific as follows: 1. synthesising probing needle, at 5 ' end, carry out sulfydryl modification, and at 3 ' end, carry out marker modification, called after SH-DNA-marker; 2. in 10 μ l 1mM SH-DNA-markers, add 0.67 μ L TCEP solution, lucifuge room temperature reaction 1h, to activate sulfydryl; 3. get Amicon-3K, the whole reaction system that adds completing steps 2. to obtain afterwards, then add 200 μ L Buffer A-1, the then centrifugal 10min of 12000rpm, with buffer A-2 washing Amicon-3K, collect the component (solution form) that molecular weight is greater than 3K; 4. get the 133 μ L 20mg/mL Streptavidin aqueous solution, add 0.33mg Sulfo-SMCC, whirlpool concussion 5min, room temperature reaction 1h then, the centrifugal 5min of 12000rpm, gets supernatant liquor; 5. get Amicon-10K, the supernatant liquor that adds step 4. to obtain, then add 200 μ L Buffer A-1, the centrifugal 10min of 12000rpm, with buffer A-2 washing Amicon-10K, collects the component (solution form) that molecular weight is greater than 10K; 6. the solution 5. step being obtained adds in the solution that 3. step obtain, lucifuge room temperature reaction 48h; 7. get Amicon-10K, the whole reaction system that adds completing steps 6. to obtain afterwards, add again 200 μ L Buffer A-1, the centrifugal 10min of 12000rpm, with buffer A-2 washing Amicon-10K, collection molecular weight is greater than the component (solution form) of 10K, is and contains the probe solution that is connected with Streptavidin and marker.TCEP solution: solute is TCEP, solvent ultrapure water, the concentration of solute is 30mM.Buffer A-1: containing the aqueous solution of 0.1M NaCl and 0.1M sodium phosphate, pH7.3.Buffer A-2: containing the aqueous solution of 0.1M NaCl, 0.1M sodium phosphate and 0.05% (volume ratio) Tween 20, pH7.3.
The preparation method of described " finishing has the magnetic bead of the aptamer of specific recognition target substance " comprises the steps: 1. 38.4mg EDC to be dissolved in the Methylimidazole damping fluid of 2mL pH 7.0,0.1M, then add aptamer described in 0.65nmol, fully mix, obtain mixed solution; 2. the mixed solution 1. step being obtained adds in about 20mg magnetic bead, room temperature shakes up reaction 24 hours, then magnetic is separated and collect magnetic bead, with 2 milliliters of prehybridization damping fluids, the unreacted carboxyl of magnetic bead surfaces is sealed, and obtains the magnetic bead that finishing has the aptamer of specific recognition target substance.Prehybridization damping fluid: solvent is the Tris damping fluid of pH7.4,0.1M, containing 0.005M EDTA, 0.5% (volume ratio) N-Sarkosyl L and 1g/100mL BSA.
The preparation method of described function magnetic bead specifically comprises the steps: 1. to get the magnetic bead that about 20mg finishing has the aptamer of specific recognition target substance, with 2mL hybridization buffer, suspends, and in 60 ℃ of water-baths, temperature is bathed 1h; 2. get 10 μ L and contain the probe solution that is connected with Streptavidin and marker, be dissolved in 2mL hybridization buffer, in 60 ℃ of water-baths, temperature is bathed 1h; 3. the solution that 2. bead suspension 1. step being obtained and step obtain mixes, and room temperature shakes up reaction 2.5h, obtains function bead suspension.Hybridization buffer: containing 10mM Tris, 120mM NaCl, 5mM KCl, 20mM CaCl
2the aqueous solution, pH 8.5.
Described marker specifically can be fluorescent marker, more specifically can be Cy5.5.The corresponding excitation wavelength of described and marker specifically can be 650nm.
Described magnetic bead specifically can be carboxyl magnetic bead.
Described solid phase chip specifically can be optical fiber.
Described target substance is Ochratoxin A.
Described aptamer is specifically as shown in the sequence 1 of sequence table.Described probe is specifically as shown in the sequence 2 of sequence table.
The object of the invention is to propose the high reusable aptamer fluorescence probe sensing analytical method of a kind of high stable.The present invention is characterized in: take aptamer as bio-identification probe, solid phase sensing, laser induced fluorescence(LIF), high stable is reused number of times.Particularly (see Fig. 1): aptamer is fixed on to magnetic bead surfaces, and hybridization the preceding paragraph complementary strand, this section of complementary dna chain puted together Streptavidin and fluorescent marker.Meanwhile, at solid phase chip modifying interface desthiobiotin.While there is target contaminant in system, complementary strand is competed, and is combined with the desthiobiotin of modifying at solid phase interface, and fluorescent substance is excited, and in the Signals & Systems of acquisition, exists target contaminant to become quantitative relationship, thereby reaches the object of detection.The inventive method measure target pollutant concentration have with low cost, simply, advantage fast, and kept that aptamers target molecules is extensive, specificity good and highly sensitive advantage, can stablize use more than 300 times.
Accompanying drawing explanation
Fig. 1 is the principle schematic of method provided by the invention.
Fig. 2 is the schematic flow sheet of the step 1 of embodiment 1.
Fig. 3 is the schematic flow sheet of the step 2 of embodiment 1.
Fig. 4 is the schematic flow sheet of the step 3 of embodiment 1.
Fig. 5 is the result of embodiment 2.
Fig. 6 is the result of embodiment 3.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.PBS damping fluid used in embodiment, if no special instructions, is the PBS damping fluid of pH7.4,10mM.Desthiobiotin: sigma, article No. is D1411.Streptavidin:: Thermo, article No. is prod#21135.Ochratoxin A: pribolab, article No. is IAC-040-3.The full name of APTS is " (3-aminopropyl) triethoxyl silane ".The Chinese full name of TCEP is " (three (2-propyloic) phosphine ", and English full name is " Tris (2-carboxyethyl) phosphine) ".The Chinese full name of Sulfo-SMCC is " 4-(N-maleimide methyl) hexanaphthene-1-carboxylic acid sulfonic group succinimide ester sodium salt ".
DNA molecular first is the aptamer of specific binding Ochratoxin A, and its nucleotide sequence (sequence 1 of sequence table) is 5-NH
2-A
6gATCGGGTGTGGGTGGCGTAAAGG
gAGCATCGGACA-3 '.DNA molecular second is the single strand dna complementary with DNA molecular first part, and its nucleotide sequence (sequence 2 of sequence table) is 5 '-AAAAAAAAAAAA
tGTCCGATGCTC-3 '.
Patent ZL200610089497.4 (CN1873450A) is shown in by evanescent wave detecting instrument (biosensor of full fiber optic evanescent wave) for the present embodiment.
The preparation of embodiment 1, test kit
One, finishing has the preparation of the optical fiber of desthiobiotin
Schematic flow sheet is shown in Fig. 2.
1, get optical fiber, make its surface hydroxylation.
Concrete grammar: (1) immerses piraha solution (98% dense H by optical fiber (570/600/900, be purchased from Nanjing Chunhui Science and Technology Industrial Co Ltd)
2sO
4: H
2o
2=3:1, volume ratio) and 120 ℃ of reaction 1h; (2) after completing steps (1), take out optical fiber, with ultrapure water, clean until pH value is neutral, at room temperature with nitrogen, dry up, be then placed in 70 ℃ of vacuum drying ovens and preserve 1h.
2, after completing steps 1, get optical fiber, make its finishing APTS.
Concrete grammar: after (1) completing steps 1, get optical fiber, be placed in APTS solution and react 1h; (2) after completing steps (1), take out optical fiber, with dry toluene, rinse, at room temperature with nitrogen, dry up, be then placed in 200 ℃ of baking 1h.APTS solution: solute is APTS, solvent is dry toluene, the concentration of solute is 2% (volume ratio).
3, after completing steps 2, get optical fiber, make its finishing desthiobiotin.
Concrete grammar: after (1) completing steps 2, get optical fiber, be placed in glutaraldehyde solution room temperature reaction 1h, then use alcohol flushing, be then placed in ethylenediamine solution room temperature reaction 1.5h, be then placed in NaBH
4room temperature reaction 15min in solution; (2) get 25mg desthiobiotin, 50mg EDC and 50mg NHS, with 1000 μ l DMF, dissolve, room temperature reaction 3h (mixing rotation), then adds the NaHCO of 200 μ l pH8.7,1M
3-Na
2cO
3damping fluid also mixes, and obtains mixed solution; (3) after completing steps (1), take out optical fiber, add in the mixed solution that step (2) obtains room temperature reaction 12 hours; (4) after completing steps (3), take out optical fiber, with alcohol flushing, be then placed in the PBS damping fluid containing 2mg/mL BSA, room temperature reaction 1h.
Glutaraldehyde solution: solute is glutaraldehyde, solvent is ethanol, the concentration of volume percent of solute is 2.5%.
Ethylenediamine solution: solute is quadrol, solvent is ethanol, the concentration of volume percent of solute is 10%.
NaBH
4solution: solute is NaBH
4, solvent is ethanol, the concentration of solute is 10mg/ml.
Two, preparation is connected with the probe of Streptavidin and fluorescent marker
Schematic flow sheet is shown in Fig. 3.
1, synthetic dna molecule second, carries out sulfydryl modification at 5 ' end, at 3 ' end, carries out Cy5.5 modification, called after SH-DNA-Cy5.5.
2, in 10 μ l 1mM SH-DNA-Cy5.5, add 0.67 μ L TCEP solution, lucifuge room temperature reaction 1h, to activate sulfydryl.TCEP solution: solute is TCEP, solvent ultrapure water, the concentration of solute is 30mM.
3, get Amicon-3K (Amicon, article No. is UFC500396-1), add the whole reaction system obtaining after completing steps 2, add again 200 μ L Buffer A-1, then the centrifugal 10min of 12000rpm, with buffer A-2 washing Amicon-3K, collect the component (solution form) that molecular weight is greater than 3K.
Buffer A-1: containing the aqueous solution of 0.1M NaCl and 0.1M sodium phosphate, pH7.3.
Buffer A-2: containing the aqueous solution of 0.1M NaCl, 0.1M sodium phosphate and 0.05% (volume ratio) Tween 20, pH7.3.
4, get the 133 μ L 20mg/mL Streptavidin aqueous solution, add 0.33mg Sulfo-SMCC, whirlpool concussion 5min, room temperature reaction 1h then, the centrifugal 5min of 12000rpm, gets supernatant liquor.
5, get Amicon-10K (Amicon, article No. is UFC501096-1), the supernatant liquor that adds step 4 to obtain, add again 200 μ L Buffer A-1, the centrifugal 10min of 12000rpm, with buffer A-2 washing Amicon-10K, collects the component (solution form) that molecular weight is greater than 10K.
6, solution step 5 being obtained adds in the solution that step 3 obtains, lucifuge room temperature reaction 48h.
7, get Amicon-10K, add the whole reaction system obtaining after completing steps 6, add again 200 μ L Buffer A-1, the centrifugal 10min of 12000rpm, with buffer A-2 washing Amicon-10K, collection molecular weight is greater than the component (solution form) of 10K, called after STV-probe-Cy5.5 solution.In Streptavidin, the concentration of STV-probe-Cy5.5 solution is 200mg/mL.
Three, the magnetic bead of preparing surperficial modification of nucleic acids aptamers
Schematic flow sheet is shown in Fig. 4.
1, get 1mL carboxyl magnetic bead (Bangs Laboratories Inc, BioMag BP618; Form of suspension, magnetic bead concentration is 20.3mg/ml), the separated and supernatant discarded of magnetic, with DEPC water washing magnetic bead 2-3 time (after each cleaning all through magnetic isolated for disposal supernatant liquor).
2,38.4mg EDC is dissolved in to the Methylimidazole damping fluid of 2mL pH 7.0,0.1M, then adds 0.65nmol DNA molecular first, fully mix, obtain mixed solution.
3, mixed solution step 2 being obtained adds in the magnetic bead that step 1 obtains, room temperature shakes up reaction 24 hours, then magnetic is separated and collect magnetic bead, with 2 milliliters of prehybridization damping fluids, the unreacted carboxyl of magnetic bead surfaces is sealed to (incubated at room 4h), obtain magnetic bead-aptamer conjugate mixed solution.
Prehybridization damping fluid: solvent is the Tris damping fluid of pH7.4,0.1M, containing 0.005M EDTA, 0.5% (volume ratio) N-Sarkosyl L and 1g/100mL BSA.
Four, the hybridization of magnetic bead-aptamer conjugate and STV-probe-Cy5.5
Schematic flow sheet is shown in Fig. 4.
1, get magnetic bead-aptamer conjugate mixed solution (containing about 20mg magnetic bead) prepared by 2mL step 3, supernatant discarded after magnetic separation, with hybridization buffer washing three times, then suspends with 2mL hybridization buffer, and in 60 ℃ of water-baths, temperature is bathed 1h.
2, get the STV-probe-Cy5.5 solution that 10 μ L step 2 obtain, be dissolved in 2mL hybridization buffer, in 60 ℃ of water-baths, temperature is bathed 1h.
3, the solution that bead suspension step 1 being obtained and step 2 obtain mixes, and room temperature shakes up reaction 2.5h, obtains function bead suspension.
Hybridization buffer: containing 10mM Tris, 120mM NaCl, 5mM KCl, 20mM CaCl
2the aqueous solution, pH 8.5.
Embodiment 2, detection Ochratoxin A
Employing DMF is solvent, the Ochratoxin A storing solution of preparation 1mg/mL.
1, get the function bead suspension (containing about 20mg magnetic bead) of embodiment 1, by the PBS buffer solution for cleaning twice containing 2mg/mL BSA, abandoning supernatant.
2, after completing steps 1, get magnetic bead, add Ochratoxin A storing solution, add the PBS damping fluid containing 2mg/mL BSA, the cumulative volume that makes reaction system is 350 μ L, mixes 15min.Ochratoxin A storing solution arranges different add-ons, the concentration that makes the initial time Ochratoxin A of reaction system is 6.4nM, 38.2nM, 95.4nM, 158.7nM, 222.6nM, 445.3nM, 635.0nM, 1269.6nM, 3174.9nM or 3710.0nM, and each concentration arranges three repetitions.
3, after completing steps 2, carry out magnetic separation, collect supernatant liquor (solution to be measured).
4, the working specification of biosensor of full fiber optic evanescent wave: open excitor, excitation wavelength is 650nm; The optical fiber direct of preparing with the step 1 of PBS damping fluid flushing embodiment 1 is steady to baseline; After baseline is steady, get the solution to be measured that step 3 obtains, open peristaltic pump sample introduction 25s, stop afterwards peristaltic pump, make solution to be measured in reaction tank, react 120s; Again open peristaltic pump, pass into elutriant (the 0.5g/100mL SDS aqueous solution is adjusted pH to 1.9) 90 seconds; Finally pass into PBS damping fluid, steady to baseline.
After the detection of a solution to be measured completes, optical fiber prepared by the step 1 of utilizing the PBS damping fluid of elutriant and pH7.4,0.1M alternately to rinse embodiment 1, the optical fiber that STV-probe-Cy5.5 is prepared from the step 1 of embodiment 1 splits away off, then carry out the detection of next solution to be measured, by this detection method, traditional DNA can be changed into the elution process of protein in conjunction with elution process, thereby reach very high stable repeated application number of times.
The results are shown in Figure 5, detecting of Ochratoxin A is limited to 12nM (4.9ng/mL), linearity range is 12nM-32 μ M.
The reused number of times of optical fiber prepared by the step 1 of embodiment 3, embodiment 1
Employing DMF is solvent, the Ochratoxin A storing solution of preparation 1mg/mL.
1, get the function bead suspension (containing about 20mg magnetic bead) of embodiment 1, by the PBS buffer solution for cleaning twice containing 2mg/mL BSA, abandoning supernatant.
2, after completing steps 1, get magnetic bead, add Ochratoxin A storing solution, add the PBS damping fluid containing 2mg/mL BSA, the cumulative volume that makes reaction system is 350 μ L (concentration of the initial time Ochratoxin A of reaction system is 3 μ M), mixes 15min.
3, after completing steps 2, carry out magnetic separation, collect supernatant liquor (solution to be measured).
4, the working specification of biosensor of full fiber optic evanescent wave: open laser apparatus, excitation wavelength is 650nm; The optical fiber direct of preparing with the step 1 of PBS damping fluid flushing embodiment 1 is steady to baseline; After baseline is steady, get the solution to be measured that step 3 obtains, open peristaltic pump sample introduction 25s, stop afterwards peristaltic pump, make solution to be measured in reaction tank, react 120s; Again open peristaltic pump, pass into elutriant (the 0.5g/100mL SDS aqueous solution is adjusted pH to 1.9) 90 seconds; Finally pass into PBS damping fluid, steady to baseline.
The solution to be measured that repeats step 3 to obtain detects.After the detection of a solution to be measured completes, optical fiber prepared by the step 1 of utilizing the PBS damping fluid of elutriant and pH7.4,0.1M alternately to rinse embodiment 1, the optical fiber that STV-probe-Cy5.5 is prepared from the step 1 of embodiment 1 splits away off, then carry out the detection of next solution to be measured, by this detection method, traditional DNA can be changed into the elution process of protein in conjunction with elution process, thereby reach very high stable repeated application number of times.
The results are shown in Figure 6.Reusable at least 300 times of the optical fiber relating in the present invention.
Claims (10)
1. detect a method that whether contains target substance in sample to be tested, comprise the steps:
(1) function magnetic bead and described sample to be tested are hatched altogether; Described function magnetic bead is hybridized and is obtained by (a) with (b); Described (a) has the magnetic bead of the aptamer of specific recognition target substance for finishing; Described (b) is for being connected with the probe of Streptavidin and marker; Described probe is the single stranded nucleic acid molecule complementary with described aptamer part;
(2) after completing steps (1), carry out magnetic separation, collect supernatant liquor;
(3), under exciting with the corresponding excitation wavelength of marker, the supernatant liquor that adopts finishing to have the solid phase chip detecting step (2) of desthiobiotin or vitamin H to obtain, judges in sample to be tested, whether to contain target substance according to signal value.
2. the method for claim 1, is characterized in that: described finishing has the preparation method of the solid phase chip of desthiobiotin to comprise the steps:
(1) get solid phase chip, make its surface hydroxylation;
(2) after completing steps (1), get solid phase chip, make its finishing APTS;
(3) after completing steps (2), get solid phase chip, make its finishing desthiobiotin.
3. method as claimed in claim 1 or 2, is characterized in that: described solid phase chip is optical fiber.
4. as the method as described in arbitrary in claims 1 to 3, it is characterized in that: described target substance is Ochratoxin A.
5. as the method as described in arbitrary in claim 1 to 4, it is characterized in that: described magnetic bead is carboxyl magnetic bead.
6. as the method as described in arbitrary in claim 1 to 5, it is characterized in that: described aptamer is as shown in the sequence 1 of sequence table, and described probe is as shown in the sequence 2 of sequence table.
7. detect a test kit that whether contains target substance in sample to be tested, comprise that function magnetic bead and finishing have the solid phase chip of desthiobiotin or vitamin H; Described function magnetic bead is hybridized and is obtained by (a) with (b); Described (a) has the magnetic bead of the aptamer of specific recognition target substance for finishing; Described (b) is for being connected with the probe of Streptavidin and marker; Described probe is the single stranded nucleic acid molecule complementary with described aptamer part.
8. detect a test kit that whether contains target substance in sample to be tested, comprise " solid phase chip that finishing has desthiobiotin or vitamin H ", " finishing has the magnetic bead of the aptamer of specific recognition target substance " and " being connected with the probe of Streptavidin and marker "; Described probe is the single stranded nucleic acid molecule complementary with described aptamer part.
9. test kit as claimed in claim 7 or 8, is characterized in that: described solid phase chip is optical fiber; Described magnetic bead is carboxyl magnetic bead.
10. as the method as described in arbitrary in claim 7 to 9, it is characterized in that: described target substance is Ochratoxin A; Described aptamer is as shown in the sequence 1 of sequence table, and described probe is as shown in the sequence 2 of sequence table.
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|---|---|---|---|---|
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101915830A (en) * | 2010-07-19 | 2010-12-15 | 江南大学 | Ochratoxin A fluorescence detection test strip and application thereof |
| CN102645420A (en) * | 2012-04-17 | 2012-08-22 | 王利兵 | Method for detecting multiple harmful substance residual components in food based on surface plasma resonance technique |
| CN103808701A (en) * | 2013-09-18 | 2014-05-21 | 河南省农业科学院 | Homogeneous-phase rapid detection method of ochratoxin A based on nucleic acid chimeric dye fluorescence quenching |
-
2014
- 2014-07-25 CN CN201410360025.2A patent/CN104178568B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101915830A (en) * | 2010-07-19 | 2010-12-15 | 江南大学 | Ochratoxin A fluorescence detection test strip and application thereof |
| CN102645420A (en) * | 2012-04-17 | 2012-08-22 | 王利兵 | Method for detecting multiple harmful substance residual components in food based on surface plasma resonance technique |
| CN103808701A (en) * | 2013-09-18 | 2014-05-21 | 河南省农业科学院 | Homogeneous-phase rapid detection method of ochratoxin A based on nucleic acid chimeric dye fluorescence quenching |
Non-Patent Citations (5)
| Title |
|---|
| BONEL L等: "An electrochemical competitive biosensor for ochratoxin A based on a", 《BIOSENS BIOELECTRON》, vol. 26, no. 7, 15 March 2011 (2011-03-15), pages 3254 - 3259, XP028148407, DOI: doi:10.1016/j.bios.2010.12.036 * |
| HUN X等: "Signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A", 《BIOSENS BIOELECTRON》, vol. 39, no. 1, 15 January 2013 (2013-01-15), pages 145 - 151 * |
| WU S等: "Aptamer-functionalized magnetic nanoparticle-based bioassay for the detection of ochratoxin A using upconversion nanoparticles as labels", 《ANALYST》, vol. 136, no. 11, 7 June 2011 (2011-06-07), pages 2306 - 2314 * |
| YANG C等: "Aptamer-DNAzyme hairpins for biosensing of Ochratoxin A", 《BIOSENS BIOELECTRON》, vol. 32, no. 1, 15 February 2012 (2012-02-15), pages 208 - 212, XP028440359, DOI: doi:10.1016/j.bios.2011.12.011 * |
| 刘腾飞等: "核酸适体的筛选制备及分析应用", 《生物技术通报》, vol. 2013, no. 4, 31 December 2013 (2013-12-31), pages 39 - 48 * |
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