CN106198503B - A kind of electrochemiluminescence sandwich biosensor and preparation and application - Google Patents
A kind of electrochemiluminescence sandwich biosensor and preparation and application Download PDFInfo
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- CN106198503B CN106198503B CN201610521857.7A CN201610521857A CN106198503B CN 106198503 B CN106198503 B CN 106198503B CN 201610521857 A CN201610521857 A CN 201610521857A CN 106198503 B CN106198503 B CN 106198503B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
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- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
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Abstract
The present invention relates to a kind of electrochemiluminescence sandwich biosensor and its preparation and application, comprising the following steps: first prepares gold nano-graphene modified glass-carbon electrode using potentiostatic method；Ru (bpy) is prepared again3 2+The beacon Ru-TBA2-AuNPs of label；Gold nano-graphene modified glass-carbon electrode is immersed containing 2~8 h are cultivated in mercapto-modified TBA1 solution, then cultivates 20 min in containing thrombin solution；20 min are finally cultivated in Ru-TBA2-AuNPs.Electrochemiluminescence sandwich biosensor of the invention is mainly used in the detection of fibrin ferment.Sensor of the invention is with good stability, reproducibility, has very strong specificity to fibrin ferment, is not easily susceptible to the interference of other substances in test sample；The features such as high sensitivity, detection quick, easy to operate.There is certain help to the research and development of blood coagulation class drug.
Description
Technical field
The present invention relates to analytical chemistry and electrochemical luminescence sensor analysis field, and in particular to one kind is based on gold nano-
The electrochemical luminescence biosensor and preparation method of graphene composite material and the detection fibrin ferment of aptamers and application.
Background technique
Fibrin ferment is primarily referred to as coagulation cascade effect protein enzyme.It is substantially carried out recycled fibre proteinogen, and to be converted into fiber white
Then body polymerize fibroblast cells, i.e. thrombus cellulose.The fibrin ferment of the mankind is mainly used for helping blood coagulation when there is wound.
The necessity of detection thrombin amount in blood not only for those by the patient of dysfunction of blood coagulation disease, and
In order to examine therapeutic agent in the effect of the processing of surgical operation and thrombus disease.Therefore, sensitive, accurate fibrin ferment detection
Method is crucial and useful in disease prevention.
Aptamers are some natural single stranded DNAs or RNA.They are folded into second level and tertiary structure, enable fibrin ferment
Enough very efficiently and specifically to combine some target molecules (small molecule, nucleic acid, protein or entire cell).Aptamers
Usually pass through the evolution index concentration ligand (SELEX) of system.With traditional recognition element --- compared with antibody, aptamers have perhaps
More unique for example the smallest immunogenicities of feature, synthesis is convenient, be easy chemical modification, stable structure is flexible.These features
Aptamers are made to become a kind of ideal candidate for developing the analysis method based on aptamers.These methods include electrochemistry
Luminescence method (ECL), electrochemical process, fluorescence method and colorimetric method.Up to the present, it is had been obtained there are two types of thrombin aptamer
It is widely applied, one is the fibrinogen sites that TBA1 is capable of bind thrombin, and binding constant Kd is in 100 nM or so.Separately
One is the better bind thrombin of TBA2 (nM of Kd=0.5).
It is compared with other methods, the advantages of Electrochemiluminescince combines electrochemistry and chemoluminescence method, such as low back
Scape signal is easy to control and low detection limit.In conjunction with the feature a little with the specificity of aptamers of electrochemical luminescence, aptamers
Electrochemical luminescence sensor has had changed into a kind of important promising detection method.Due to Ru (bpy)3 2+/ TprA system goes out
The stability of color and very high luminous efficiency, Ru (bpy)3 2+/ TprA system has obtained good research.Especially based on raising
Ru(bpy)3 2+In-depth study has been done in terms of/TprA system luminescent properties.Nano material such as carbon nanotube, graphene, Jenner
The application that rice and nanocomposite are used to amplify electrochemical luminescence signals obtains in electrochemical luminescence sensor
It is widely applied.The nanocomposite based on graphene obtains nearest graphene in building electronic component
It is extensive to pay attention to, as in Laser emission, solar battery.These electronic components require the conduction of some frivolous and electronics mostly
High-efficient material.Graphene is a kind of graphite material of monatomic thickness, and graphene-metallic composite possesses remarkably
The such as big specific surface area of physicochemical properties, controllable electronic property.These properties allow graphene-metallic composite to exist
Have become promising electrode material in building electrochemical luminescence sensor.
Summary of the invention
The purpose of the present invention is to provide a kind of electrochemiluminescence sandwich biosensors and its preparation and application to solve
At present to the detection device of fibrin ferment and complex disposal process, it is expensive, sensitivity is not high the problems such as.
A kind of preparation method of electrochemiluminescence sandwich biosensor, mainly comprises the steps that
(S1) gold nano-graphene modified glass-carbon electrode is prepared using potentiostatic method；
(S2) AuNPs is prepared using citric acid reduction gold chloride method；
(S3) Ru (bpy) is used2(dcbpy) NHS marks TBA2 and is dissolved in PBS solution, obtains Ru-TBA2 beacon；
(S4) by Ru-TBA2 beacon TCEP(trichloroethyl phosphate obtained by step (S3)) and pH be 5.2 acetate delay
It rushes solution and pre-processes 1 h to disconnect the cystine linkage formed between sulfydryl；
(S5) AuNPs obtained by step (S2) is added in the processed vial of NaOH, is added obtained by step (S4)
Ru-TBA2 is added Tris- acetate buffer solution and NaCl that pH is 8.2 after cultivating 16 h under room temperature shading, continues
24 h are cultivated under greenhouse shading；It is centrifuged off extra Ru-TBA2；It is molten that obtained Ru-TBA2-AuNPs is dissolved into PBS buffering
In liquid；
(S6) gold nano-graphene modified glass-carbon electrode obtained by the step (S1) is immersed containing mercapto-modified
2~8 h are cultivated in TBA1 solution, then cultivate 20 min in containing thrombin solution；The finally Ru- obtained by step (S5)
20 min are cultivated in TBA2-AuNPs.
Gold nano-graphene composite material, the good nature that existing graphene has, unique structure make it have excellent
Electricity, calorifics, mechanics and chemical property, and under the interaction of gold nano, these properties have apparent enhancing.And
And gold nano is capable of providing the site of sulfydryl modification DNA molecular modification.In the present invention, the gold nano of electrode surface and sulfydryl are repaired
The aptamers TBA1 of decorations can effective fixed adaptation body TBA1 by Au-S chemical bonds.Another aspect gold nano-graphite
Alkene composite material can substantially increase the electronics conduction efficiency of electrode, can thus increase the response of electrochemical luminescence signals.
Ru(bpy)3 2+It is a kind of stable, very high luminescence reagent of luminous efficiency, due to its molecular weight and molecule space
Volume is much smaller compared with the label such as enzyme, and does not influence the specificity and cross reactivity of nucleic acid, so that Ru (bpy)3 2+On a molecular scale
Nucleic acid is marked and is possibly realized.Along with Ru (bpy)3 2+Regeneration cycle reaction can be carried out in electrochemical luminescence reaction,
So that a marker can generate a considerable amount of photons in each detection cycle, thus sensitivity for analysis is very high.At this
Ru (bpy) is utilized in the method for invention3 2+Label TBA2 not only will not influence the combination of aptamers TBA2 and fibrin ferment, but also
The sensitivity of detection reaction can also be enhanced.In the present invention, in the solution, Ru is modified with using gold nano grain connection
(bpy)3 2+TBA2, since to can connect the TBA2 molecules of many quantity a kind of radial to be formed for a gold nano grain
Electrochemical luminescence signals source (Ru-TBA2-AuNPs), that will be introduced a large amount of when occurring a small amount of fibrin ferment in system
Luminescence reagent is so further exaggerated the response of electrochemical luminescence signals.
The electrochemical luminescence biosensor prepared through the invention, due to Ru (bpy)3 2+It marks on aptamers TBA2,
And then TBA2 and gold nano form the signal macromolecular of Ru-TBA2-AuNPs.Aptamers TBA1 is connected to by Au-S chemical bond
It is modified with gold nano-graphene electrode surface.When occur in system blood coagulation it is mould when, fibrin ferment will be tied with TBA1 and TBA2
It closes, so that Ru-TBA2-AuNPs is introduced into electrode surface, is realized by the enhancing of electrochemical luminescence signals to fibrin ferment
Quantitative detection.
Biomolecule needs certain electrolyte environment, and sodium chloride mainly provides electrolyte environment.
Further, the mercapto-modified TBA1 sequence is 5 '-SH- (CH2)6-TTTTTGGTTGGTGTGGTTGG-3'；
The sequence of the Ru-TBA2 beacon is 5 '-SH- (CH2)6-TTTTTTTTTTTTTTTAGTCCGTAGGGCAGGTGGGGGG
TGACTT-(CH2)6-NH2-3’。
Further, the citric acid reduction gold chloride method preparation AuNPs, which is specifically included that, takes chlorauric acid solution in container
It is heated to boiling, is then quickly added into sodium citrate；Continue to heat and ceaselessly stir until the color of solution is become by yellow
Red, then ceaselessly agitating solution is cooled to room temperature, is stored in spare under 4 °C.
Further, potentiostatic method preparation gold nano-graphene modified glass-carbon electrode is specifically included that using oxygen
Graphite alkene and gold chloride are potentiostatic electrodeposition under -1.1 V to 600~1000 s of glass-carbon electrode in current potential.Use glass-carbon electrode
It is preceding first by glass-carbon electrode α-A12O3Polishing powder polishing；It washs, respectively 10 min of ultrasound in deionized water and ethyl alcohol.
Further, the Ru (bpy)2(dcbpy) synthesis of NHS mainly comprises the steps that
(S11) by Ru (bpy)2Cl2, sodium bicarbonate and 2,2 '-bipyridyl -4,4 '-dicarboxylic acids be uniformly mixed, be added ethyl alcohol -
Cold filtration preparation Ru (bpy) after heated in water solution reflux2(dcbpy)(PF6)2；
(S12) again by N, DMF solution is added after being stirred in N '-dicyclohexylcarbodiimide and n-hydroxysuccinimide
Make it that Ru (bpy) be added after completely dissolution2(dcbpy)(PF6)2Middle synthesis Ru (bpy)2(dcbpy)NHS。
Further, the concentration of step (S5) preparation gained Ru-TBA2-AuNPs is 2~8 μM；Step (S2) is prepared into
The AuNPs partial size arrived is 6-10 nm.If Ru-TBA2-AuNPs concentration is too low, in the electrochemical luminescence letter that electrode surface introduces
Number source is very little, influences the response of electrochemical luminescence signals, such as excessively too many, will increase the steric hindrance of sensor surface, also unfavorable
In the introducing of signal source.
Further, the pH value of the PBS buffer solution is 7.4.
A kind of electrochemiluminescence sandwich biosensor that above-mentioned preparation method obtains.
A kind of application of above-mentioned electrochemiluminescence sandwich biosensor, is mainly used for the detection of fibrin ferment.Due to blood coagulation
Enzyme is capable of combination the aptamers TBA1 and TBA2 of specificity, so in the case where blood coagulation mould appearance, luminescence reagent Ru-TBA2-
AuNPs may be introduced into electrode surface, so as to cause the enhancing of electrochemical luminescence signals, according to the increasing of electrochemical luminescence signals
Strong degree realizes the quantitative detection to fibrin ferment.Electrochemical luminescence biosensor is limited to 6.3 pM to the detection of fibrin ferment.
Further, the detection of the fibrin ferment mainly comprises the steps that
(S21) the electrochemiluminescence sandwich biosensor is immersed in 20~40 min in thrombin solution to be measured,
Then electrode is rinsed with phosphate buffer solution；
(S22) the electrochemiluminescence sandwich biosensor is immersed in 20 in 1~10 μM of Ru-TBA2-AuNPs~
Then 40 min rinse electrode with phosphate buffer solution；
(S23) using the electrochemical luminescence biosensor as working electrode in three-electrode system, with 0.05~0.1
M Tri-n-Propylamine solution carries out electrochemistry and electrochemical luminescence detection as detection solution.
Compared with prior art, the electrochemical luminescence biosensor of detection fibrin ferment of the invention has the advantages that
1) with good stability, reproducibility has very high specificity to fibrin ferment, is not easily susceptible to other substances in test sample
Interference；2) raising of the gold nano-graphene composite material to electronics conduction efficiency is utilized, the sound of electrochemical luminescence quotation marks is promoted
It answers, and amplification of the introducing to electrochemical luminescence signals of big signal source Ru-TBA2-AuNPs, to greatly increase this hair
The sensitivity of bright sensor；3) simple to operate, can quick and precisely in test sample fibrin ferment content.Electricity of the invention
Thrombin amount of the chemiluminescence biosensor in detection blood embodies good performance, therefore to blood coagulation class drug
Research and development have certain help.
Detailed description of the invention
Fig. 1 is that the electrochemical luminescence biosensor of invention is used to detect the experimental principle figure of fibrin ferment；
Fig. 2 is cyclic voltammogram of the glass-carbon electrode of invention in different phase in the 5 mM potassium ferricyanides/potassium ferrocyanide；
Fig. 3 is electrochemical impedance of the glass-carbon electrode in different phase of invention；
Fig. 4 is the relationship of the biosensor luminous intensity and Au-TBA2-AuNPs cultivation time in fibrin ferment of invention
It is bent；
Fig. 5 be invention biosensor luminous intensity in various concentration fibrin ferment 0.01nM (a), 0.05nM
(b), the relation curve of time is cultivated in 0.1 nM (c)；
Fig. 6 is gold nano-graphene shape appearance figure；
Fig. 7 is the electrochemical luminescence signals response of the fibrin ferment of the biosensor detection various concentration of invention, electrochemistry
The canonical plotting of luminous intensity and concentration of thrombin；
Variation before and after the electrochemical luminescence signals two weeks of the biosensor detection fibrin ferment of Fig. 8 invention；
Fig. 9 is biosensor anti-interference figure of the invention.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing
Step ground detailed description.
Embodiment 1
A kind of preparation method for the electrochemical luminescence biosensor detecting fibrin ferment, as shown in Figure 1, mainly including following
Step:
(1) pretreatment of glassy carbon electrode: glass-carbon electrode is through 0.05 μm of α-A12O3After polishing powder polishing, rinsed with deionized water
Completely, and respectively 10 min of ultrasound in deionized water and ethyl alcohol.It is detected using three-electrode system, working electrode is glass carbon electricity
Pole is platinum electrode to electrode, and reference electrode is Ag/AgCl electrode, and in 0.5 M sulfuric acid, setting voltage is -0.2~1.6
V carries out cyclic voltammetry scan to glass-carbon electrode, after detection, rinses electrode with deionized water, dries up electrode surface, spare.
(2) gold nano-graphene modified glass-carbon electrode preparation: being 0.6 mg mL in concentration-1Graphene oxide and 25
The gold chloride of mM, 600 s of potentiostatic electrodeposition in the case where current potential is -1.1 V.Wherein graphite oxide is managed according to improved Hummers
It is prepared by by crystalline flake graphite.Gold nano-graphene the shape appearance figure being prepared is as shown in fig. 6, as can be seen from Figure 6 have perhaps
More gold nano grains are distributed on the surface of graphene, and gold nano grain is uniform in size, and the composite structure illustrated is equal
It is even.
(3) preparation of illuminated beacon Ru-TBA2-AuNPs solution:
1) gold nano grain and synthesis (AuNPs)
AuNPs is obtained by the method for restoring gold chloride with citric acid.It is specific as follows, take 50 mL, 1~5 mM's
Chlorauric acid solution ebuillition of heated in conical flask, then the sodium citrate of 5 mL, 30~40 mM is quickly added in solution.
Solution continues ebuillition of heated and ceaselessly stirs, and the color of solution becomes red by yellow.Ceaselessly agitating solution is cooled to room
Temperature has thus obtained AuNPs colloidal solution, is stored in spare under 4 °C.
2) synthesis of Ru-TBA2
① Ru(bpy)2(dcbpy)(PF6)2Synthesis: weigh the Ru (bpy) of i.e. 0.328 mM of 0.16 g2Cl2、0.16
2,2 '-bipyridyl -4 of g sodium bicarbonate and i.e. 0.492 mM of 0.12 g, 4 '-dicarboxylic acids uniformly mix, and 10 mL volume ratios are added
To be heated to reflux 4h after the ethanol-water solution of 4:1；After the completion of back flow reaction, the cooling 7h of ice bath, in cooling procedure, using 1 M salt
Acid solution adjusts reaction solution pH to 4.4 to promote to react the crystalline deposit for generating Chinese red and be precipitated；By the reaction after sufficient crystallising
Liquid is filtered, and obtained crystallization is filtered to remove insoluble impurity after being dissolved again with methanol, obtains bright Chinese red
Filtrate；By the sodium hexafluoro phosphate (NaPF of 14 mL6) (2 g) solution is added in Chinese red filtrate, the cooling 7h of ice bath guarantees to produce
Object being capable of complete Precipitation；Using the resulting sediment of micro-pore-film filtration, and it is dried in vacuo, it is finally obtained red
Color pulverulent solids are Ru (bpy)2(dcbpy)(PF6)2。
②Ru(bpy)2(dcbpy) synthesis of NHS: by the 0.46 g i.e. N of 2.22 mM, N '-dicyclohexylcarbodiimide
(DCC) it is stirred with 0.238 g i.e. 2.08 mM n-hydroxysuccinimides (NHS), 3 mL is added under condition of ice bath
DMF solution dissolves it sufficiently；Lysate stirring is added to 1 ml and contains 0.38 g Ru (bpy)2(dcbpy)(PF6)2's
In DMF solution, continue to stir 5h under normal temperature condition so that active carboxyl functional group Ru (bpy) 2 (dcbpy) is finally prepared
NHS DMF solution；A small amount of reaction mixture is taken to be added to progress ultraviolet spectra characterization in the PBS solution of 0.10 M, according to it 279
Occurs characteristic peak and previous experiments work at nm and 453 nm, conversion estimated concentration is 6.02 × 10-4 M。
3. single-stranded b form dna ruthenium complex label: Ru (bpy)2(dcbpy) NHS can be used directly label 3 ' and hold amido
The TBA2(of change its aptamers TBA2:5 '-SH- (CH2)6-TTTTTTTTTTTTTTTAGTCCGTAGGGCAGGTGGGGGG
TGACTT-(CH2)6-NH2- 3 ').Firstly, 200 μ L is taken to dissolve Ru (bpy)2(dcbpy) PBS solution of NHS is added to containing
In the centrifuge tube of 5 mL of 200 μ L TBA2 solution (1OD), low speed concussion 12h is protected from light under the conditions of 25 DEG C of constant temperature；After the completion of concussion,
The sodium acetate solution of 100 μ L, 3 M and 2mL ice dehydrated alcohol are added to reaction system to terminate and reacted, it is quiet under the conditions of -20 DEG C
Set 12h；After the completion of standing, reaction mixture 30 min of low-temperature centrifugation under 12000 r/min revolving speeds carefully removes supernatant,
It is rinsed sediment 2 times with 70% ice dehydrated alcohol again, is centrifuged 10 min after having rinsed every time, finally washed away not by DNA combination
Ru(bpy)2(dcbpy)NHS；Reaction precipitate after freeze-drying is the electrochemical luminescence beacon after tris (bipyridine) ruthenium label
Ru-TBA2。
3) synthesis of Ru-TBA2-AuNPs
The synthesis of Ru-TBA2-AuNPs, the TCEP and 1 μ L 500 of Ru-TBA2 1.5 μ L, 10 mM of 9 μ L, 1 mM
The acetate buffer solution of pH=5.2 mM pre-processes 1 h to disconnect the cystine linkage formed between sulfydryl.Then the AuNPs of 3 mL
It is added to in the processed vial of NaOH, then above-mentioned Ru-TBA2 is added in vial, is cultivated under room temperature shading
16 h.The NaCl of 1 M of the Tris- of pH=8.2 acetate buffer solution and 300 μ L of 30 μ L, 500 mM is slowly added into body
In system, mixed liquor continues to cultivate 24 h under greenhouse shading.16000 r/min centrifugation, 25 min are removed extra at room temperature
Ru-TBA2.4.035 μ g Ru-TBA2-AuNPs are finally taken to be dissolved into the PBS buffer solution of 0.1 pH=7.4 M 200 μ L
It is configured to 2 μM of Ru-TBA2-AunNPS solution, is stored in spare under 4 °C.
(4) building of biosensor
1. be modified with gold nano-graphene composite material glass-carbon electrode be immersed in 20 μ L containing mercapto-modified
2 h of TBA1 solution.
2. electrode is then cultivated 20 min in containing thrombin solution.
3. cultivating 20 min above-mentioned electrode being immersed in the Ru-TBA2-AuNPs containing 2 μM of 20 μ L.
The present embodiment in order to study modified glassy carbon electrode biosensor detection blood coagulation of the invention electrode reaction property,
Mechanism and Kinetics parameters of electrode processes.Pretreated glass-carbon electrode is recycled using three-electrode system and is lied prostrate before being modified
Peace method is tested.
Detection method mainly comprises the steps that
(1) i.e. by working electrode be pretreated glass-carbon electrode, to electrode be platinum electrode, reference electrode Ag/
The three-electrode system of AgCl electrode carries out cyclic voltammetry scan to glass-carbon electrode, is provided with voltage in 0.5 M sulfuric acid
For -0.2~1.6 V, after detection, glass-carbon electrode is rinsed with deionized water, dries up glassy carbon electrode surface, it is spare.
(2) electrode is impregnated into fibrin ferment and Ru-TBA2-AuNPs 20 min, then uses phosphate-buffered respectively
Solution rinses electrode.
(3) modified electrode that preparation is completed is put into 0.05 M Tri-n-Propylamine solution and carries out cyclic voltammetry scan, wherein
Wherein voltage range is 0.2~1.25 V, and sweep speed is 100 mV s-1, photomultiplier tube high pressure is set as -800 V
Embodiment 2
A kind of preparation method for the electrochemical luminescence biosensor detecting fibrin ferment, comprising the following steps:
(1) pretreatment of glassy carbon electrode: glass-carbon electrode is through 0.05 μm of α-A12O3After polishing powder polishing, rinsed with deionized water
Completely, and respectively 10 min of ultrasound in deionized water and ethyl alcohol.It is detected using three-electrode system, working electrode is glass carbon electricity
Pole is platinum electrode to electrode, and reference electrode is Ag/AgCl electrode, and in 0.5 M sulfuric acid, setting voltage is -0.2~1.6
V carries out cyclic voltammetry scan to glass-carbon electrode, after detection, rinses electrode with deionized water, dries up electrode surface, spare.
(2) gold nano-graphene modified glass-carbon electrode preparation: being 0.8 mg mL in concentration-1Graphene oxide and 55
The gold chloride of mM, 1000 s of potentiostatic electrodeposition in the case where current potential is -1.1 V.Wherein graphite oxide is managed according to improved Hummers
It is prepared by by crystalline flake graphite.Gold nano-graphene the shape appearance figure being prepared is as shown in fig. 6, as can be seen from Figure 6 have perhaps
More gold nano grains are distributed on the surface of graphene, and gold nano grain is uniform in size, and the composite structure illustrated is equal
It is even.
(3) preparation of illuminated beacon Ru-TBA2-AuNPs solution: the preparation process of Ru-TBA2-AuNPs with embodiment 1,
It takes the Ru-TBA2-AuNPs of 16.14 μ g to rinse to be dissolved in the PBS solution of 200 μ L, is configured to the Ru- that concentration is 8 μM
TBA2-AuNPs solution.And it is saved backup under the conditions of 4 DEG C.
(4) building of biosensor
1. be modified with gold nano-graphene composite material glass-carbon electrode be immersed in 20 μ L containing mercapto-modified
8 h of TBA1 solution.
2. electrode is then cultivated 40 min in containing thrombin solution.
3. cultivating 40 min above-mentioned electrode being immersed in the Ru-TBA2-AuNPs containing 8 μM of 20 μ L.
The present embodiment in order to study modified glassy carbon electrode biosensor detection blood coagulation of the invention electrode reaction property,
Mechanism and Kinetics parameters of electrode processes.Pretreated glass-carbon electrode is recycled using three-electrode system and is lied prostrate before being modified
Peace method is tested.
Detection method mainly comprises the steps that
(1) i.e. by working electrode be pretreated glass-carbon electrode, to electrode be platinum electrode, reference electrode Ag/
The three-electrode system of AgCl electrode carries out cyclic voltammetry scan to glass-carbon electrode, is provided with voltage in 0.5 M sulfuric acid
For -0.2~1.6 V, after detection, glass-carbon electrode is rinsed with deionized water, dries up glassy carbon electrode surface, it is spare.
(2) electrode is impregnated into fibrin ferment and Ru-TBA2-AuNPs 40 min, then uses phosphate-buffered respectively
Solution rinses electrode.
(3) modified electrode that preparation is completed is put into 0.1M Tri-n-Propylamine solution and carries out cyclic voltammetry scan, wherein its
Middle voltage range is 0.2~1.25 V, and sweep speed is 100 mV s-1, photomultiplier tube high pressure is set as -800 V.
Embodiment 3
1, cyclic voltammetry compares
Following processing a is respectively according to the glass-carbon electrode that the processing of embodiment 1 or 2 obtains for 5 to be modified with for bare electrode, b
Gold nano-graphene composite material electrode, c are modified with TBA1, d and introduce fibrin ferment, e introducing TBA2-Ru-AuNPs.Again
A, the cyclic voltammetry of b, c, d, e in the 5 mM potassium ferricyanides/potassium ferrocyanide, test results are shown in figure 2, only works as gold
After nanometer-graphene composite material modified electrode, electric current has apparent increase, and it is good to illustrate that composite material of the invention has
Electric conductivity.
2, electrochemical impedance test comparison
5 are handled according to embodiment 1 or 2 and obtains glass-carbon electrode and be following processing a respectively being modified with gold for bare electrode, b
Electrode, the c of nanometer-graphene composite material are modified with TBA1, d and introduce fibrin ferment, e introducing TBA2-Ru-AuNPs.Again a,
B, c, d, e carry out testing impedance, impedance spectra that test results are shown in figure 3, as can be seen from the figure when gold nano-graphene is repaired
Impedance has apparent reduce to further illustrate that composite material has good electric conductivity after decorations electrode.
3, the research of time is cultivated in Au-TBA2-AuNPs
Gold nano-graphene modified the glass-carbon electrode obtained according to embodiment 1 or 2 is immersed containing mercapto-modified
2~8 h are cultivated in TBA1 solution, then cultivate 20 min in containing thrombin solution.Finally according to the present embodiment 1 or 2
The different time is cultivated in obtained Ru-TBA2-AuNPs, as shown in figure 4, a, b and c be respectively concentration be 0.01nM,
The fibrin ferment of the concentration of 0.05nM and 0.1 nM cultivates the change of the electrochemical luminescence signals of different time in Ru-TBA2-AuNPS
Change, as can be seen from the figure electrochemical luminescence signals are continuously increased as time increases, are reached stable in 20 min, are illustrated
It is 20 min that the time is most preferably cultivated in Ru-TBA2-AuNPS.
4, the research of time is cultivated in fibrin ferment
Gold nano-graphene modified the glass-carbon electrode obtained according to embodiment 1 or 2 is immersed containing mercapto-modified
2~8 h are cultivated in TBA1 solution, then cultivate different time in containing thrombin solution.Finally according to the present embodiment 1 or
Cultivate 20min in 2 obtained Ru-TBA2-AuNPs, as shown in figure 5, a, b and c be respectively concentration be 0.01nM, 0.05nM and
The fibrin ferment of 0.1 nM electrochemical luminescence signals change curve in the different cultivation times, as can be seen from Figure 5 with the time
Increase electrochemical luminescence signals be continuously increased, reach stable in 30 min, illustrate that most preferably cultivating the time in fibrin ferment is
30 min。
5, the influence that concentration responds electrochemical luminescence signals in fibrin ferment
It is respectively the solidifying of 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM and 10 nM by the concentration of a-g
Hemase is detected according to the method for embodiment 1 or embodiment 2, as shown in fig. 7, the electrochemistry of the fibrin ferment to various concentration
The logarithm of response intensity and concentration of thrombin is in a linear relationship, and linear relationship is fine.
6, sensor stability is tested
As shown in figure 8, a is the electrochemical luminescence signals before two weeks, b is the luminous signal of two weeks, can be with from figure
Signal intensity is little before and after finding out two weeks, illustrates that sensor is very stable.
7, selective test
In order to probe into sensor to the anti-interference of other substances, to other common substance bovine serum albumins, lysozyme,
Human albumin, immunoglobulin, insulin, trypsase, histidine carry out parallel interference experiment.It can be seen in figure 9 that
Sensor has strong signal to respond fibrin ferment, to other interfering substances almost without signal response.The experimental results showed that this
The sensor of embodiment preparation has good interference free performance, has good fibrin ferment identification specificity.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, although referring to aforementioned reality
Applying example, invention is explained in detail, for those skilled in the art, still can be to aforementioned each reality
Technical solution documented by example is applied to modify or equivalent replacement of some of the technical features.It is all of the invention
Spirit and principle within, any modification, equivalent replacement, improvement and so on, should be included in protection scope of the present invention it
It is interior.
SEQUENCE LISTING
<110>University Of Shantou
<120>a kind of electrochemiluminescence sandwich biosensor and preparation and application
<130> 2016
<160> 2
<170> PatentIn version 3.3
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<211> 24
<212> DNA
<213>artificial sequence
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<212> DNA
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<223> n is a, c, g, or t
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Claims (9)
1. a kind of preparation method of electrochemiluminescence sandwich biosensor, which is characterized in that mainly comprise the steps that
(S1) gold nano-graphene modified glass-carbon electrode is prepared using potentiostatic method；
(S2) AuNPs is prepared using citric acid reduction gold chloride method；
(S3) Ru (bpy) is used2(dcbpy) NHS marks TBA2 and is dissolved in PBS solution, obtains Ru-TBA2 beacon；
(S4) acetate buffer solution that TCEP and pH is 5.2 of Ru-TBA2 beacon obtained by step (S3) is pre-processed into 1h；
(S5) AuNPs obtained by step (S2) is added in the processed vial of NaOH, adds Ru- obtained by step (S4)
Tris- acetate buffer solution and NaCl that pH is 8.2 is added after cultivating 16h under room temperature shading in TBA2, continues to hide in greenhouse
It is cultivated for 24 hours under light；It is centrifuged off extra Ru-TBA2；Obtained Ru-TBA2-AuNPs is dissolved into PBS buffer solution；
(S6) gold nano-graphene modified glass-carbon electrode obtained by the step (S1) is immersed and contains mercapto-modified TBA1
2~8h is cultivated in solution, then cultivates 20min in containing thrombin solution；The finally Ru-TBA2- obtained by step (S5)
20min is cultivated in AuNPs；
Wherein, the mercapto-modified TBA1 sequence is 5 '-SH- (CH2)6-TTTTTGGTTGGTGTGGTTGG-3'；
The sequence of the Ru-TBA2 beacon is 5 '-SH- (CH2)6-TTTTTTTTTTTTTTTAGTCCGTAGGGCAGGTGGGGGG
TGACTT-(CH2)6-NH2-3’。
2. preparation method according to claim 1, which is characterized in that the citric acid reduction gold chloride method prepares AuNPs master
It include: that chlorauric acid solution is taken, to boiling, to be then quickly added into sodium citrate in vessel in heating；Continue to heat and ceaselessly stir
It mixes the color until solution and becomes red by yellow, then ceaselessly stirring allows solution to be cooled to room temperature, is stored in spare at 4 DEG C.
3. preparation method according to claim 1, which is characterized in that the potentiostatic method prepares gold nano-graphene modified
Glass-carbon electrode specifically include that using graphene oxide and gold chloride, potentiostatic electrodeposition is to glass-carbon electrode in the case where current potential is -1.1V
600~1000s.
4. preparation method according to claim 1, which is characterized in that the Ru (bpy)2(dcbpy) synthesis of NHS is mainly wrapped
Include following steps:
(S11) by Ru (bpy)2Cl2, sodium bicarbonate and 2,2 '-bipyridyl -4,4 '-dicarboxylic acids be uniformly mixed, be added alcohol-water it is molten
Rear cold filtration preparation Ru (bpy) is heated to reflux in liquid2(dcbpy)(PF6)2；
(S12) again by N, DMF solution, which is added, after being stirred in N '-dicyclohexylcarbodiimide and n-hydroxysuccinimide makes it
Ru (bpy) is added after completely dissolution2(dcbpy)(PF6)2Middle synthesis Ru (bpy)2(dcbpy)NHS。
5. preparation method according to claim 1, which is characterized in that step (S5) preparation gained Ru-TBA2-AuNPs's is dense
Degree is 2~8 μM；The AuNPs partial size that step (S2) is prepared is 6-10nm.
6. preparation method according to claim 1, which is characterized in that the pH of the PBS buffer solution is 7.4.
7. a kind of electrochemiluminescence sandwich biosensor that preparation method obtains according to claim 1.
8. the application of electrochemiluminescence sandwich biosensor according to claim 7, which is characterized in that be mainly used for blood coagulation
The detection of enzyme.
9. applying according to claim 8, which is characterized in that the detection of the fibrin ferment mainly comprises the steps that
(S21) the electrochemiluminescence sandwich biosensor is immersed in 20~40min in thrombin solution to be measured, then used
Phosphate buffer solution rinses electrode；
(S22) the electrochemiluminescence sandwich biosensor is immersed in 20 in 1~10 μM of Ru-TBA2-AuNPs~
Then 40min rinses electrode with phosphate buffer solution；(S23) using the electrochemical luminescence biosensor as work electricity
Pole carries out electrochemistry and electrochemical luminescence inspection using 0.05~0.1M Tri-n-Propylamine solution as detection solution in three-electrode system
It surveys.
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| CN104062330B (en) * | 2014-06-11 | 2016-05-18 | 汕头大学 | A kind of electrochemical luminescence biology sensor that detects fibrin ferment and preparation method thereof |
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