CN109738495A - Three metal signals amplification aptamer sensor based on ce metal organic frame@golden nano-complexes and golden platinum ruthenium nanocomposite is detected for thrombin antithrombin III complex - Google Patents
Three metal signals amplification aptamer sensor based on ce metal organic frame@golden nano-complexes and golden platinum ruthenium nanocomposite is detected for thrombin antithrombin III complex Download PDFInfo
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Abstract
The present invention uses aptamers as target acquistion agent, signal based on ce metal organic frame@golden nano-complexes (Ce-MOF@Au) and golden platinum ruthenium nanocomposite (AuPtRu NP) amplifies strategy, successfully develops the sensor for thrombin antithrombin III complex (TSP-1) Sensitive Detection.The AuPtRu NP of synthesis acts not only as the catalyst of catalyzing hydrogen peroxide, is also used as nano-carrier capture amino (- NH2) and terminates single stranded DNA (S1) to obtain signal probe SP (AuPtRu NP/S1).Ce-MOF@Au is obtained by in-situ reducing and is used as the electrode modification material of glass-carbon electrode.When detection solution contains H2O2Electrochemistry matrix when, AuPtRu NP can aoxidize H2O2To obtain the signal of enhancing.The invention shows go out from 1fg mL‑1To 10ng mL‑1The range of linearity, have 0.13fg mL‑1Low-down detection limit.In addition, this aptamer sensor is proved to be used directly for the detection of clinical practice sample.
Description
Technical field:
The present invention relates to a kind of clinically electrochemical aptamer sensors of quantitative detection thrombin antithrombin III complex
Preparation method and application, is based especially on ce metal organic frame@golden nano-complexes (Ce-MOF@Au) and golden platinum ruthenium nanometer is multiple
Condensation material (AuPtRu NP) belongs to electricity for detecting thrombin antithrombin III complex as biosensor prepared by signal probe
Field of chemical detection.
Background technique:
Cardiovascular disease (CVD) is the disease of heart and blood vessel, and is the main reason for whole world is dead.With CVD wind
The individual of danger may show the increase of biomarker molecular level in blood.Identification is used for the highest CVD wind of auxiliary diagnosis
The biomarker of danger can prevent premature death.Thrombospondin-1 (TSP-1) is that a kind of promising CVD candidate is raw
Object marker is expressed higher in large-scale atherosclerotic lesion and myocardial infarction.It therefore, it has been developed to a series of sides
Method, such as Tetrazolium salt colorimetric assay and enzyme-linked immunosorbent assay (ELISA), for detecting TSP-1.However, all these sides
Method requires to carry out complex process to target sample.Therefore, the sensitive and selective enumeration method of TSP-1 still has in clinical sample
Challenge.Due to the potentiality of its extremely low detection limit and in-situ study, electrochemical sensor be can be realized in CVD diagnosis
TSP-1 detection.
In recent years, organic using metal ion as node and organic ligand as the novel microporous material-metal of connector
Frame (MOFs), gradually causes the concern of researchers.MOF has adjustable size and form, has high porosity, makes us full
The electrochemical stability of meaning and easily modified feature.By MOFs spy's sexual enlightenment, synthesize in this work based on cerium
(Ce) the advantages of MOF (Ce-MOF), it not only inherits traditional MOF, but also show powerful delivered payload capability and good
Biocompatibility.The advantages of Ce-MOF, shows that Ce-MOF is highly suitable as electrode modification material.We have found that with equal benzene three
Formic acid has the advantages that high efficiency and step are simple as organic backbone synthesis Ce-MOF, this will undoubtedly increase sensor building
Feasibility.In order to increase sensor sensitivity and immobilized more capture probe DNA (CP), by gold nano grain (AuNPs)
It modifies on the surface of the Ce-MOF of preparation to serve as nano-carrier, amido modified CP is fixed by gold-ammonia key.At us
In pervious work, it has been found that using reducing agent by golden in-situ reducing arrive carrier surface when be easy assemble, this makes synthesis side
Method is extremely limited.It is reported that PVP molecule can be used as typical SURFACTANT ADSORPTION on the noble metal core of growth, prevent simultaneously
The only aggregation of noble metal nano particles.In order to solve this problem, this work in, polyvinylpyrrolidone (PVP) by with
Make protective agent, successfully avoids the aggregation of AuNPs.As far as we know, this is to develop one-step method for the first time to go back Au NP in situ
It is former to form stable nanocomposite (Ce-MOF@Au) to the surface Ce-MOF and be used as electrode modification material.
Due to the unstable disadvantage of traditional biological identification molecule (such as: enzyme, antibody), them are limited in bio-sensing
The use of device.And aptamers have high-affinity and the specificity to the various target substances from small molecule to large protein,
It is increasingly used in various sensing platforms.Aptamers are the short synthesis obtained by the in-vitro method from cell SELEX
Nucleotide sequence.Other than they are to the affinity of target, aptamers, which also have, to be such as readily produced, low cost synthesis, high
Thermal stability and be easy to mark and modify the advantages of.They can also be easily fixed at the difference with various chemical property
On surface.Therefore, the substitute in various sensors as antibody is applied to using aptamers as specific recognition substance.We
Target be design it is a kind of by aptamers specificity with the aptamer sensor that signal amplification mechanism combines be used for TSP-1 examine
It surveys.
In order to realize the Sensitive Detection of trace materials, secondary singal substance analyzes skill commonly used in amplification electrochemical sensor
Electrochemical response in art.Platinum (Pt) nano particle has a good catalytic performance for a long time, and reasonable composition and
Structure is most important for design high activity platinum catalyst.Currently, having synthesized more and more a variety of three metal materials of platinum, example
Such as there is FePtCu, AuPdPt and the PtPdTe of catalytic action, their catalytic capability is all an advantage over pair of individual platinum and platinum
Metal material.In addition, can be used as a kind of outstanding catalyst it is understood that metal Ru (Ru) is highly stable.Also,
Ruthenium is effective curing agent of platinum, can be used for improving the stability and catalytic performance of platinum.Au NPs has good chemical stability
With good biocompatibility, it is widely used in electrochemical signals material.Therefore, this research combines Au NPs, Pt NPs
The advantages of with Ru NPs, design has synthesized AuPtRu tri-metal nano compound (AuPtRu NPs) for the first time.AuPtRu NP has
The ability of good stability, catalytic performance and fixing biological molecules.In brief, by AuPtRu NPs to H2O2Collaboration
Catalysis, electrochemical signals have obtained greatly enlarged.
In order to construct multicomponent, synergic nano sensing interface designs the amino (- NH for having modification in 5 ' ends2) core
Single-stranded 1 (S1) of acid reacts to form double-strand with aptamers in PCR machine.Then, S1 passes through Au-NH2Key and Pt-NH2Key and letter
Number materials A uPtRu NPs connection is formed signal probe (SP).In the presence of TSP-1, TSP-1 is in conjunction with aptamers and induces
SP is discharged from M1 (SP+ aptamers) are free.Meanwhile by-NH2The capture probe (CP) of modification is fixed on to be modified by Ce-MOF@Au
Electrode on.When CP identifies the SP of release and captures them, AuPtRu is catalyzed H2O2Generate big electric signal.Due to Ce-MOF@
Au has the excellent catalytic capability of excellent electric conductivity and AuPtRu NPs, and new technical platform is realized to TSP-1 in serum
Sensitive Detection.We demonstrate the aptamer sensors proposed to have a good application prospect in clinical studies.
The project, which establishes a simple, quick detection method, realizes special, super sensitivity detection to TSP-1.For
Cardiovascular patient early detection and risk profile provide foundation.
Summary of the invention:
1. the purpose of the present invention is the preparation sides of the electrochemical aptamer sensor for detecting thrombin antithrombin III complex
Method and application, provide foundation for clinically cardiovascular patient early detection and risk profile, feature the following steps are included:
(1) ce metal organic frame@golden nano-complexes (Ce-MOF@Au) and golden platinum ruthenium nanocomposite (AuPtRu
NPs) the preparation of signal probe；
(2) electrochemical aptamer sensor is established, thrombin antithrombin III complex is detected, draws standard curve.
2. ce metal organic frame@golden nano-complexes of the present invention and golden platinum ruthenium nanocomposite signal probe
Preparation process specifically includes following steps, feature the following steps are included:
(1) preparation of Ce-MOF@Au composite material:
Firstly, by the Ce (NO of 4.34g3)3·6H2O is added in the ultrapure water of 45mL, and dissolution forms solution A.Then with
10mL water-ethyl alcohol (1: 1) makees solvent, and the H of 2.10g is added3BTC forms solution B.By solution A 60 DEG C, be vigorously stirred
It is gradually added dropwise in solution B under conditions of (800rpm), sufficiently reaction one hour.Be centrifugated (8000rpm), with ethyl alcohol and
Milli-Q water for several times, obtains Ce-MOF, drying for standby.The preparation for then carrying out nanocomposite Ce-MOF@Au, claims first
It takes the Ce-MOF of 1mg to be dispersed in the ultrapure water of 2mL, 2mL 2%HAuCl is then added4, it is added 2mL's after mixing well
PVP solution (2mg mL-1), ultrasound mixes 15min.It stirs at 800 rpm, 2mL NaBH is slowly added dropwise4(7.5mg mL-1), room
Temperature continues to stir 30min, centrifugation, and ultrapure washing three times, obtains Ce-MOF Au nano-complex, and be dispersed in Milli-Q water
It further uses.
(2) preparation of golden platinum ruthenium Tri-metal nanoparticle (AuPtRu NPs):
Firstly, by 0.9mL 20mM RuCl3, 53.2 μ L 5%HAuCl4, 88.4uL 5%H2PtCl6And 0.01g
Pluronic F127 is placed in beaker, mixes.Then (800rpm) is gradually added dropwise to 0.3mL 0.4M AA under stiring, at room temperature
Continue stirring 3 hours.Centrifugation, ultrapure washing three times, are dispersed in 500uL ultrapure water.
(3) preparation of signal probe (SP):
After 4 μM of signal chains (S1) are mixed in equal volume with 4 μM of aptamers chains first, heated 10 minutes at 90 DEG C, it is then cold
But it is kept for 1 hour to room temperature, obtains the double-strand of partial hybridization.200 μ L are taken to hybridize the AuPtRu tri- that the double-strand to be formed is added to 1mL
It in metal mixed solution, is vibrated 12 hours in 4 DEG C, mixture is centrifuged, washing twice (8000rpm), is dissolved in 1mL hybridization solution
In, 0.25%BSA is added thereto later and closes nonspecific binding site, vibrates 2 hours at 4 DEG C, is centrifuged again
(5000rpm), washing is primary, is dispersed in the hybridization solution of 1mL.200 μ L are finally added into the mixing liquid of above-mentioned acquisition not
With the thrombin antithrombin III complex of concentration, the signal chains in double-strand is caused to dissociate to get off, just obtained with electro-chemical activity
Signal probe (SP).
3. according to claim 1 establish electrochemical aptamer sensor, thrombin antithrombin III complex is detected, is drawn
Standard curve, it is characterised in that the following steps are included:
(1) respectively with 0.3 and 0.05 μm of Al2O3Powder by polishing electrode at mirror surface, then respectively by ultrapure water, anhydrous
Sequence each 5min of ultrasound electrode of ethyl alcohol, ultrapure water, drying at room temperature are spare；
(2) golden (the Ce-MOF@Au) nano-complex of 8 μ L electrode modified material ce metal organic frame@is added dropwise in electrode
Surface is dried at room temperature.
(3) 4 μM of capture probe (CP) solution of 10uL are integrated to dry electrode surface (37 DEG C, 2.5h).
(4) electrode washing after incubation is completely added dropwise to 6 μ L, 0.5% BSA solution incubation at room temperature afterwards with ultrapure water
30min。
(5) after clean 10 μ L juxtaposition on the electrode is added dropwise in the signal probe prepared (SP) by electrode washing with ultrapure water
In 37 DEG C of incubation 2h.
(6) it is dry that the electrode after incubation is completely placed on to room temperature condition with ultrapure water.
(7) electrode is placed in 5mL, 0.1M PBS (0.1M Na2HPO4, 0.1M KH2PO4, 0.1M KCI) in carry out table
20 μ L, 2.2mM H are added every 50s in sign2O2, measure its chrono-amperometric variable-current value.
(8) in a linear relationship according to gained current variation value and thrombin antithrombin III complex concentration, draw working curve.
Compared with prior art, the preparation side of the electrochemical aptamer sensor of a kind of quantitative detection TSP-1 of the invention
Method and application, the feature protruded is:
(1) ce metal organic frame@golden nano-complexes (Ce-MOF@Au) is used as electrode modified material, by golden platinum ruthenium
Nanocomposite not only effectively raises the catalytic performance of material as signal probe, but also improves biomolecule
Supported quantity, and then improve the sensitivity and detection range of electrochemical aptamer sensor；
(2) it realizes and can be used for TSP-1 electrochemistry super sensitivity detection in true clinical sample.
(3) electrochemical aptamer sensor of this method preparation can for it is clinical early diagnose cardiovascular patient provide according to
According to, and can be used for the risk that prediction cardiovascular event occurs.In addition, the method is easy, quickly, it is easy to implement commodity
Change, to promote the development of accurate medicine.
(4) identical nano material and method of modifying are used, different aptamers and corresponding complementation can be used
Chain detects while to realize to various biomolecules, provides more comprehensive foundation for the diagnosis of disease.
Detailed description of the invention:
Fig. 1 is the building schematic diagram of electrochemical aptamer sensor of the invention.
Fig. 2 is ce metal organic frame of the invention and ce metal organic frame@golden nano-complexes (Ce-MOF@Au)
Compare electron microscope, DPV figure, XPS figure and UV absorption figure.
Fig. 3 is the electron microscope of golden platinum ruthenium nanocomposite of the invention, zeta potential figure and chronoamperogram.
Fig. 4 is electrode building process atomic force microscope and electrochemical Characterization of the invention.
Fig. 5 be the chrono-amperometric variable-current that is obtained when detect TSP-1 of electrochemical aptamer sensor of the invention and
The linear relationship of concentration and the reproducibility of sensor and specificity.
Specific embodiment:
The present invention is further elaborated combined with specific embodiments below, it should be appreciated that these embodiments are merely to illustrate
The present invention rather than limit the scope of the invention.
Embodiment 1
Step 1. is firstly, by the Ce (NO of 4.34g3)3·6H2O is added in the ultrapure water of 45mL, and dissolution forms solution A.So
Solvent is made with 10mL water-ethanol (1: 1) afterwards, the H of 2.10g is added3BTC forms solution B.By solution A 60 DEG C, be vigorously stirred
It is gradually added dropwise in solution B under conditions of (800rpm), sufficiently reaction one hour.Be centrifugated (8000rpm), with ethyl alcohol and
Milli-Q water for several times, obtains Ce-MOF, drying for standby.The preparation for then carrying out nanocomposite Ce-MOF@Au, claims first
It takes the Ce-MOF of 1mg to be dispersed in the ultrapure water of 2mL, 2% HAuCl of 2mL is then added4, it is added 2mL's after mixing well
PVP solution (2mg mL-1), ultrasound mixes 15min.It stirs at 800 rpm, 2mL NaBH is slowly added dropwise4(7.5mg mL-1), room
Temperature continues to stir 30min, centrifugation, and ultrapure washing three times, obtains Ce-MOF Au nano-complex, and be dispersed in Milli-Q water
In.
Step 2. is respectively with 0.3 and 0.05 μm of Al2O3Polishing electrode at mirror surface, is then pressed ultrapure water, nothing by powder respectively
Sequence each 5min of ultrasound electrode of water-ethanol, ultrapure water, drying at room temperature are spare；
8 μ L electrode modified material ce metal organic frame@golden nano-complexes (Ce-MOF@Au) are added dropwise in electricity step 3.
Pole surface is dried at room temperature.
4 μM of capture probe (CP) solution of 10uL are integrated to dry electrode surface (37 DEG C, 2.5h) by step 4..
6 μ L, 0.5% BSA solution incubation at room temperature are completely added dropwise with ultrapure water by step 5. afterwards for electrode washing after incubation
30min。
With ultrapure water 10 μ L are added dropwise on the electrode after clean in the signal probe prepared (SP) by electrode washing by step 6.
It is placed in 37 DEG C of incubation 2h.
It is dry that electrode after incubation is completely placed on room temperature condition by step 7. with ultrapure water.
Electrode is placed in 5mL, 0.1M PBS (0.1M Na by step 8.2HPO4, 0.1M KH2PO4, 0.1M KCI) in carry out
20 μ L, 2.2mM H are added every 50s in characterization2O2, measure its chrono-amperometric variable-current value；
Step 9. is in a linear relationship according to gained current variation value and thrombin antithrombin III complex (TSP-1) concentration, draws
Working curve；Measurement result shows TSP-1 concentration in 1fg mL-1-10ng mL-1It is linear in range, linear correlation system
Number is 0.9975, and detection is limited to 0.13fg mL-1。
Step 11. saves the sensor of the present invention in 4 DEG C, the response of discontinuity detection sensor current, after storage 30 days
Current-responsive is still the 91.60% of initial current, indicates that sensor has excellent stability；
Step 12. present invention takes immunosensor 5 of same batch preparation, under the same conditions to 100pg mL-1's
TSP-1 is measured respectively, each determination of electrode 3 times, sensor reproducibility is excellent.
Step 13. will detect TSP-1 under the conditions of existing for the sensor of the present invention in blood other biological molecule, tie
The presence of fruit other biological molecule does not influence the change of TSP-1 electric current, illustrates that the specificity of sensor is good, can distinguish mesh very well
Mark detectable substance.
The above is only a preferred embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, under the precondition for not departing from the principle of the invention, several improvements and modifications can also be made, these improve and
Retouching also should be regarded as protection scope of the present invention.
Claims (3)
1. three metal signals based on ce metal organic frame@golden nano-complexes and golden platinum ruthenium nanocomposite amplify adaptation
Body sensor is detected for thrombin antithrombin III complex, it is characterised in that the following steps are included:
(1) preparation of golden (the Ce-MOF@Au) nano-complex of ce metal organic frame@and golden platinum ruthenium (AuPtRu NP) three metals
The preparation of signal probe；
(2) electrochemical aptamer sensor is established, is detected thrombin antithrombin III complex (TSP-1), standard curve is drawn.
2. the preparation of golden (the Ce-MOF@Au) nano-complex of ce metal organic frame@and golden platinum ruthenium (AuPtRu NP) three metals letter
The preparation process of number probe, feature the following steps are included:
(1) preparation of Ce-MOF@Au composite material:
Firstly, by the Ce (NO of 4.34g3)3·6H2O is added in the ultrapure water of 45mL, and dissolution forms solution A.Then with 10mL water-
Ethyl alcohol (1: 1) makees solvent, and the H of 2.10g is added3BTC forms solution B.By solution A 60 DEG C, be vigorously stirred the item of (800rpm)
It is gradually added dropwise in solution B under part, sufficiently reaction one hour.It is centrifugated (8000rpm), with ethyl alcohol and milli-Q water number
It is secondary, obtain Ce-MOF, drying for standby.The preparation for then carrying out nanocomposite Ce-MOF@Au, weighs the Ce- of 1mg first
MOF is dispersed in the ultrapure water of 2mL, and 2mL 2%HAuCl is then added4, the PVP solution (2mg of 2mL is added after mixing well
mL-1), ultrasound mixes 15min.It stirs at 800 rpm, 2mL NaBH is slowly added dropwise4(7.5mg mL-1), room temperature continues to stir
30min, centrifugation, ultrapure washing three times, obtain Ce-MOF Au nano-complex, and be dispersed in Milli-Q water and further make
With.
(2) preparation of golden platinum ruthenium Tri-metal nanoparticle (AuPtRu NPs):
Firstly, by 0.9mL 20mM RuCl3, 53.2 μ L 5%HAuCl4, 88.4uL 5%H2PtCl6And 0.01g
Pluronic F127 is placed in beaker, mixes.Then (800rpm) is gradually added dropwise to 0.3mL 0.4MAA under stiring, at room temperature
Continue stirring 3 hours.Centrifugation, ultrapure washing three times, are dispersed in 500uL ultrapure water.
(3) preparation of signal probe (SP):
After 4 μM of signal chains (S1) are mixed in equal volume with 4 μM of aptamers chains first, heats 10 minutes, be subsequently cooled at 90 DEG C
Room temperature is kept for 1 hour, obtains the double-strand of partial hybridization.200 μ L are taken to hybridize tri- metal of AuPtRu that the double-strand to be formed is added to 1mL
In mixed solution, to be vibrated 12 hours in 4 DEG C, mixture is centrifuged, washing twice (8000rpm), is dissolved in 1mL hybridization solution, it
0.25%BSA is added thereto afterwards and closes nonspecific binding site, is vibrated at 4 DEG C 2 hours, is centrifuged (5000rpm) again, water
It washes once, is dispersed in the hybridization solution of 1mL.The blood coagulation of 200 μ L various concentrations is finally added into the mixing liquid of above-mentioned acquisition
Enzyme sensitive Protein -1, causes the signal chains in double-strand to dissociate to get off, and just obtains the signal probe (SP) with electro-chemical activity.
3. according to claim 1 establish electrochemical aptamer sensor, thrombin antithrombin III complex is detected, draws standard
Curve, it is characterised in that the following steps are included:
(1) respectively with 0.3 and 0.05 μm of Al2O3Powder by polishing electrode at mirror surface, then respectively by ultrapure water, dehydrated alcohol,
Sequence each 5min of ultrasound electrode of ultrapure water, drying at room temperature are spare；
(2) golden (the Ce-MOF@Au) nano-complex of 8 μ L electrode modified material ce metal organic frame@is added dropwise in electrode surface,
It dries at room temperature.
(3) 4 μM of capture probe (CP) solution of 10uL are integrated to dry electrode surface (37 DEG C, 2.5h).
(4) electrode washing after incubation is completely added dropwise to 6 μ L afterwards with ultrapure water, 0.5% BSA solution is incubated at room temperature 30min.
(5) after clean 10 μ L are added dropwise in the signal probe prepared (SP) by electrode washing with ultrapure water and are placed in 37 on the electrode
DEG C be incubated for 2h.
(6) it is dry that the electrode after incubation is completely placed on to room temperature condition with ultrapure water.
(7) electrode is placed in 5mL, 0.1M PBS (0.1M Na2HPO4, 0.1M KH2PO4, 0.1M KCl) in characterized, every
20 μ L, 2.2mM H are added in 50s2O2, measure its chrono-amperometric variable-current value.
(8) in a linear relationship according to gained current variation value and thrombin antithrombin III complex concentration, draw working curve.
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