CN107607501A - A kind of biomarker multiple detection method based on fluorescent quenching - Google Patents
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Abstract
A kind of biomarker multiple detection method based on fluorescent quenching, belongs to field of biological detection.The present invention includes：Carboxyl quantum dot modifies aptamers DNA molecular, and golden nanometer particle modifies the complementary series of adaptor molecules, the formation of fluorescent quenching system and the measure of fluorescence signal.The present invention is by means of the quantum dot of three kinds of different emissions of three kinds of different biomarker aptamers modifications as fluorescence probe, pass through aptamers and the recognition reaction of biomarker, so that not with the aptamers that biomarker combines by with three kinds of corresponding aptamers complementary sequence hybridizations being modified on golden nanometer particle, it is quenched so as to cause golden nanometer particle to produce the fluorescence of quantum dot, by three kinds of quantum dot fluorescence signal intensities in measure system, so as to realize the detection to every kind of biomarker.This method is simple, convenient, high sensitivity, specificity it is good, the Multiple detection of biomarker can be realized, a kind of efficient detection method is provided for the quick diagnosis of disease.
Description
Technical field
A kind of biomarker multiple detection method based on fluorescent quenching, belongs to field of biological detection.
Background technology
Biomarker reference table aggregate structure and functional status or reflection poisonous substance cause the biomedical effect such as body injury
The recognizable material answered.Biomarker has very high the presence or absence of specificity, biomarker and changes of contents and body work(
The occurrence and development of energy state, disease have close relationship, and biomarker is detected as crowd's screening, clinical diagnosis, treatment
Effect, Observation On The Prognosis evaluation bring strong analysis foundation, therefore, detection to biomarker and medical diagnosis on disease during
Extremely important effect in performance.
Protein is a kind of important biomarker, many Tumor biomarkers belong to enzyme, Antigens material and
The protein matters such as microglobulin, ferritin.Currently used method of protein detection includes radioimmunoassay experiment（RIA）, it is enzyme-linked
Immunoadsorption assay（ELISA）, protein immunization imprinting the methods of, although these methods can realize protein detection and quantitative,
But it is restricted in the highly sensitive detection field of some needs.Therefore the detection method for developing highly sensitive biomarker is non-
It is often important.
Aptamers are the Fas lignand system evolution technologies by index concentration（SELEX）One kind of screening can identify target point
The one section of single stranded DNA or RNA molecule of son, are a kind of new biological identification molecules, and the identification of this quasi-molecule and target molecule has
There are high-affinity and high specific, and be obtained with by synthesizing in vitro, a large amount of quickly preparations can be carried out, fitted simultaneously
The stability of ligand molecular is small by the interference of environmental factor, and stability is stronger, therefore is widely used in field of biosensors.
When aptamer and target substance are specifically bound, the configuration of aptamer itself can change therewith, will
Aptamer is applied to the sensor that identification probe can develop a variety of change of configuration based on aptamer.
Quantum dot is made of semi-conducting material (being generally made up of the A of IIB ~ VI or IIIA ~ VA elements), stable diameter
In 2-20 nm nano-particle.Because electronics and hole become have molecular characterization by quantum confinement, continuous band structure
Discrete energy level structure, fluorescence can be launched after being excited.Simple quantum dot particle is easily influenceed by impurity and lattice defect,
But when using it as core, being wrapped up with another semi-conducting material, after forming core-shell structure, it can not only strengthen the stabilization of product
Property and dispersiveness, can also by quantum yield improve 50%, the quantum dot of different-grain diameter can also be obtained from ultraviolet to near-infrared model
Enclose the spectrum of interior arbitrfary point.Quantum dot is with a wide range of applications in biomedicine field as biological fluorescent labeling, because
Quantum dot has excellent spectral signature and photochemical stability, can be bigger widen quantum dot as bioprobe in biology
The application of detection field.
The present invention modifies the quantum dot of three kinds of different emissions the aptamers of three kinds of different biomarkers respectively, fits
The recognition reaction of part and biomarker causes aptamers structure to change, by its adaptation corresponding with being modified with three kinds
The golden nanometer particle mixing of body complementary series, it is in pairs that the aptamers sequence not combined with biomarker is complementary to sequence hybridization
Chain structure, so as to cause golden nanometer particle to produce quenching effect to the fluorescence of quantum dot, after being reacted by measure three kinds in system
The change of the fluorescence signal of quantum dot, so as to obtain the corresponding pass between every kind of biomarker and corresponding fluorescence intensity
System, so as to be detected respectively to every kind of biomarker.
The content of the invention
Technical problems to be solved：Existing biological marker object detecting method has needed highly sensitive detection field at some
Through being difficult to reach detection demand, so as to limit the use range of existing method.Meanwhile the detection of single creature mark can not
The quick comprehensive information that disease is provided, it is therefore desirable to develop multiple detection method to improve the detection efficiency of disease.
Technical scheme：A kind of biomarker multiple detection method based on fluorescent quenching, comprises the following steps：
(1) carboxyl quantum dot modification aptamers DNA molecular
Three kinds of different launch wavelength intervals are more than to 50nm carboxyl quantum dot（Have purchased from the exploitation of Wuhan Ka source technology of quantum dots
Limit responsible company）Modify the adaptor molecules with amino of three kinds of biomarkers respectively with carbodlimide method, measured with one kind
Exemplified by son point, concretely comprise the following steps：Quantum dot is diluted to 10nM concentration with pH7.4 0.01M phosphate buffer, to 200
1mg carbodiimides and 2mg N- hydroxy thiosuccinimides are added in μ L50nM quantum dot solution, is shaken at ambient temperature
Reaction 1h is swung, then carries out ultrafiltration, then the phosphate buffer with pH7.4 0.01M with the super filter tube that molecular cut off is 1000
It is resuspended, so as to obtain the quantum dot of aptamers modification.
(2) complementary series of golden nanometer particle modification adaptor molecules
By the particle diameter newly synthesized be 15-20nm golden nanometer particle under conditions of 8000r/min 5 times of centrifugal concentrating, measure gold
The concentration of nano-particle is 10nM, will be with step（1）In three kinds of complementary DNA molecule mixed in equal amounts corresponding to three kinds of aptamers, will
It is added in 200 μ L 10nM golden nanometer particle so that the final concentration of 200nM of every kind of DNA molecular, DNA molecular and Jenner
Rice corpuscles is with 20:1 mol ratio carries out modification reaction, after being incubated 6h at ambient temperature, by golden nanometer particle in 9000r/min
Under conditions of centrifugation to remove unnecessary DNA molecular.
(3) measure of the formation of fluorescent quenching system and fluorescence signal
By step（1）In obtain aptamers modification three kinds of quantum dots mix in equal volume, be dispensed into PCR pipe, often the μ L of pipe 50；
By three kinds of biomarker molecule mixed in equal amounts, then 10 concentration are diluted to, specific concentration is：0.01pg/mL、
0.05pg/mL, 1pg/mL, 5pg/mL, 10pg/mL, 20pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, so
The biomarker molecule 10uL of above various concentrations is often separately added into pipe quantum dot solution backward, is vibrated at ambient temperature
React 30-60min；The reacted μ L of solution 20 are taken to be added separately to step（2）100 μ L gold nanoparticle probes of middle preparation are molten
In liquid, after being incubated 1h at ambient temperature, three kinds of fluorescence signal intensities of often pipe are determined, and establish biomarker molecular concentration
With the corresponding relation between corresponding fluorescence signal intensity.
Three kinds of quantum used in a kind of biomarker multiple detection method based on fluorescent quenching of the present invention
The launch wavelength of point is respectively 505nm, 585nm, 645nm.
The three kinds of biologies detected in a kind of biomarker multiple detection method based on fluorescent quenching of the present invention
Marker molecules are PSA, MUC1, fibrin ferment.
Prostate specific resists in a kind of biomarker multiple detection method based on fluorescent quenching of the present invention
Original, MUC1, the aptamers sequence of fibrin ferment and corresponding complementary series are：
PSA aptamers DNA1：5’-NH2-ATTAAAGCTCGCCATCAAATAGC-3’。
DNA1 complementary series：5’-SH-ATGGCGAGCTTTAAT-3’.
Mucin aptamers DNA2：5’-NH2-GCAGTTGATCCTTTG GATACCCTGG-3’。
DNA2 complementary series：5’- SH-CAAAGGATCAACTGC-3’.
Thrombin aptamer DNA3：5’-NH2-GGTTGGTGTGGTTGG-3’。
DNA3 complementary series：5’-SH-CCAACCACACCAACC-3’.
Jenner's grain of rice used in a kind of biomarker multiple detection method based on fluorescent quenching of the present invention
The particle diameter of son is 20nm.
The amount that aptamers are modified in a kind of biomarker multiple detection method based on fluorescent quenching of the present invention
The reaction time of son point and biomarker is 45min.
The method that 20nm of the present invention golden nanometer particle reduces gold chloride by trisodium citrate is synthesized, and is closed
Into step：By there-necked flask chloroazotic acid soaked overnight, then cleaned up with ultra-pure water, add 96mL's in the there-necked flask of cleaning
Ultra-pure water, the gold chloride that 2.5mL mass concentrations are 0.4%, magnetic agitation and ebuillition of heated are added, 1.8 are added after 7-8min
ML mass concentrations are 1% trisodium citrate, and solution is changed into stopping heating after red, continues to stir 15min, that is, obtain from colourless
20nm golden nanometer particles.
Beneficial effect：The quantum dot of three kinds of different emissions is modified three kinds of different biomarkers by the present invention respectively
The recognition reaction of aptamers, aptamers and biomarker causes aptamers structure to change, by it with being modified with three kinds of phases
The golden nanometer particle mixing of corresponding aptamers complementary series, the aptamers sequence not combined with biomarker are complementary to sequence
Row are hybridized to duplex structure, so as to cause golden nanometer particle to produce quenching effect to the fluorescence of quantum dot, after determining reaction
The change of the fluorescence signal of three kinds of quantum dots in system, so as to obtain between every kind of biomarker and corresponding fluorescence intensity
Corresponding relation, so as to being detected respectively to every kind of biomarker.The method is simple to operate, while can realize more
The detection of kind biomarker, is a kind of preferable multiple detection method.
Brief description of the drawings
The fluorescence spectra of tri- kinds of quantum dots of Fig. 1.
The fluorescence value changes of Fig. 2 PSAs detection.
The fluorescence value changes of Fig. 3 mucins detection.
The fluorescence value changes of Fig. 4 fibrin ferments detection.
Embodiment
Embodiment 1
A kind of biomarker multiple detection method based on fluorescent quenching, comprises the following steps：
（1）Carboxyl quantum dot modifies aptamers DNA molecular
The carboxyl quantum dot that three kinds of launch wavelengths are respectively 505nm, 585nm, 645nm is modified three kinds with carbodlimide method respectively
Biomarker（PSA, mucin, fibrin ferment）The adaptor molecules with amino, with a kind of quantum dot
Exemplified by, concretely comprise the following steps：Quantum dot is diluted to 10nM concentration with pH7.4 0.01M phosphate buffer, to 200 μ
1mg carbodiimides and 2mg N- hydroxy thiosuccinimides are added in L50nM quantum dot solution, is vibrated at ambient temperature
1h is reacted, then carries out ultrafiltration with the super filter tube that molecular cut off is 1000, then will with pH7.4 0.01M phosphate buffer
It is resuspended, so as to obtain the quantum dot of aptamers modification.
PSA aptamers DNA1：5’-NH2-ATTAAAGCTCGCCATCAAATAGC-3’。
Mucin aptamers DNA2：5’- GCAGTTGATCCTTTG GATACCCTGG-3’.
Thrombin aptamer DNA3：5’-GGTTGGTGTGGTTGG-3’.
（2）Golden nanometer particle modifies the complementary series of adaptor molecules
By the new golden nanometer particle for synthesizing 20nm 5 times of centrifugal concentrating under conditions of 8000r/min, the dense of golden nanometer particle is measured
Spend for 10nM, will be with step（1）In three kinds of complementary DNA molecule mixed in equal amounts corresponding to three kinds of aptamers, add it to 200 μ L
In 10nM golden nanometer particle so that the final concentration of 200nM of every kind of DNA molecular, DNA molecular is with golden nanometer particle with 20:1
Mol ratio carries out modification reaction, after being incubated 6h at ambient temperature, golden nanometer particle is centrifuged under conditions of 9000r/min with
Remove unnecessary DNA molecular.
DNA1 complementary series：5’-SH-ATGGCGAGCTTTAAT-3’.
DNA2 complementary series：5’- SH-CAAAGGATCAACTGC-3’.
DNA3 complementary series：5’-SH-CCAACCACACCAACC-3’.
（3）The measure of fluorescence signal and biomarker sensitivity analysis
By step（1）In obtain aptamers modification three kinds of quantum dots mix in equal volume, be dispensed into PCR pipe, often the μ L of pipe 50；
By three kinds of biomarker molecule mixed in equal amounts, then 10 concentration are diluted to, specific concentration is：0.01pg/mL、
0.05pg/mL, 1pg/mL, 5pg/mL, 10pg/mL, 20pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, so
The biomarker molecule 10uL of above various concentrations is often separately added into pipe quantum dot solution backward, is vibrated at ambient temperature
React 45min；The reacted μ L of solution 20 are taken to be added separately to step（2）100 μ L gold nanoparticle probe solution of middle preparation
In, after being incubated 1h at ambient temperature, three kinds of fluorescence signal intensities of the every pipe of measure, and establish biomarker molecular concentration with
Corresponding relation between corresponding fluorescence signal intensity；And the range of linearity for thus measuring PSA detection exists
Between 0.01-20pg/mL, the range of linearity of mucin detection is between 1-100pg/mL, the 5-100pg/mL of fibrin ferment detection
Between.The detection sensitivity that PSA is calculated is 0.0048pg/mL, and the detection sensitivity of mucin is
0.62pg/mL, the detection sensitivity of fibrin ferment is 2.4pg/mL.
（4）Selectivity and the actual sample analysis of detection method
In the HSA containing 500nM（Human albumin）Human serum sample in, add PSA, glutinous egg respectively
In vain, content 0.05pg/mL, 1pg/mL, 10pg/mL of thrombin standard product, 1pg/mL, 5pg/mL, 25pg/mL, 2pg/mL,
8pg/mL, 40pg/mL, 5pg/mL, 15pg/mL, 60pg/mL, 10pg/mL, 30pg/mL, 80pg/mL, prostate specific resist
Former TIANZHU XINGNAO Capsul is 91.2-95.8%, and the TIANZHU XINGNAO Capsul of mucin is 93.8-97.9%, the TIANZHU XINGNAO Capsul of fibrin ferment
For 90.7-96.7%, so as to show that detection of the method to biomarker is not disturbed by sample, TIANZHU XINGNAO Capsul, which meets, to be added
The detection range that add-back is received.
Sequence table
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Claims (6)
1. a kind of biomarker multiple detection method based on fluorescent quenching, it is characterised in that comprise the following steps：
Carboxyl quantum dot modifies aptamers DNA molecular
Carboxyl quantum dot of three kinds of different launch wavelength intervals more than 50nm is modified into three kinds of biologies with carbodlimide method respectively
The adaptor molecules with amino of mark, by taking a kind of quantum dot as an example, are concretely comprised the following steps：With pH7.4 0.01M phosphoric acid
Quantum dot is diluted to 10nM concentration by salt buffer, into 200 μ L50nM quantum dot solution add 1mg carbodiimides and
2mg N- hydroxy thiosuccinimides, oscillating reactions 1h at ambient temperature, then with the ultrafiltration that molecular cut off is 1000
Pipe carries out ultrafiltration, then is resuspended with pH7.4 0.01M phosphate buffer, so as to obtain the quantum dot of aptamers modification；
Golden nanometer particle modifies the complementary series of adaptor molecules
By the particle diameter newly synthesized be 15-20nm golden nanometer particle under conditions of 8000r/min 5 times of centrifugal concentrating, measure gold
The concentration of nano-particle is 10nM, will be with step（1）In three kinds of complementary DNA molecule mixed in equal amounts corresponding to three kinds of aptamers, will
It is added in 200 μ L 10nM golden nanometer particle so that the final concentration of 200nM of every kind of DNA molecular, DNA molecular and Jenner
Rice corpuscles is with 20:1 mol ratio carries out modification reaction, after being incubated 6h at ambient temperature, by golden nanometer particle in 9000r/min
Under conditions of centrifugation to remove unnecessary DNA molecular；
The formation of fluorescent quenching system and the measure of fluorescence signal
By step（1）In obtain aptamers modification three kinds of quantum dots mix in equal volume, be dispensed into PCR pipe, often the μ L of pipe 50；
By three kinds of biomarker molecule mixed in equal amounts, then 10 concentration are diluted to, specific concentration is：0.01pg/mL、
0.05pg/mL, 1pg/mL, 5pg/mL, 10pg/mL, 20pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, so
The biomarker molecule 10uL of above various concentrations is often separately added into pipe quantum dot solution backward, is vibrated at ambient temperature
React 30-60min；The reacted μ L of solution 20 are taken to be added separately to step（2）100 μ L gold nanoparticle probes of middle preparation are molten
In liquid, after being incubated 1h at ambient temperature, three kinds of fluorescence signal intensities of often pipe are determined, and establish biomarker molecular concentration
With the corresponding relation between corresponding fluorescence signal intensity.
A kind of 2. biomarker multiple detection method based on fluorescent quenching according to claim 1, it is characterised in that
The launch wavelength of three kinds of described quantum dots is respectively 505nm, 585nm, 645nm.
A kind of 3. biomarker multiple detection method based on fluorescent quenching according to claim 1, it is characterised in that
Three kinds of described biomarker molecules are PSA, MUC1, fibrin ferment.
A kind of 4. biomarker multiple detection method based on fluorescent quenching according to claim 3, it is characterised in that
Described PSA, MUC1, the aptamers sequence of fibrin ferment and corresponding complementary series be：
PSA aptamers DNA1：5’-NH2-ATTAAAGCTCGCCATCAAATAGC-3’；
DNA1 complementary series：5’-SH-ATGGCGAGCTTTAAT-3’；
Mucin aptamers DNA2：5’-NH2-GCAGTTGATCCTTTG GATACCCTGG-3’；
DNA2 complementary series：5’- SH-CAAAGGATCAACTGC-3’；
Thrombin aptamer DNA3：5’-NH2-GGTTGGTGTGGTTGG-3’；
DNA3 complementary series：5’-SH-CCAACCACACCAACC-3’.
A kind of 5. biomarker multiple detection method based on fluorescent quenching according to claim 1, it is characterised in that
The particle diameter of described golden nanometer particle is 20nm.
A kind of 6. biomarker multiple detection method based on fluorescent quenching according to claim 1, it is characterised in that
Described step（3）The quantum dot of middle aptamers modification and the reaction time of biomarker are 45min.
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