CN109490284A - A kind of dual catalytic Particles in Electrochemiluminescofce ofce Luminol biosensor based on gold nano grain and two titanium carbide MXenes - Google Patents
A kind of dual catalytic Particles in Electrochemiluminescofce ofce Luminol biosensor based on gold nano grain and two titanium carbide MXenes Download PDFInfo
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Abstract
This disclosure relates to a kind of dual catalytic Particles in Electrochemiluminescofce ofce Luminol biosensor based on gold nano grain and two titanium carbide MXenes, in conjunction with multidigit point recognition strategy and New Two Dimensional material-Ti3C2The reproducibility of MXenes forms AuNPs-MXenes as ECL signal probe, designs highly sensitive electrochemical luminescence sensing platform to detect Hela excretion body.In the strategy, SA polymer molecule can provide multidigit point identification interface to capture more aptamers to improve the capture rate of excretion body, this facilitates the sensitivity of improvement biosensor and AuNP-MXenes can be with dual catalytic luminol ECL signal enhancing.Based on both dual amplification strategies, highly sensitive and highly selective biosensor is obtained to detect excretion body, in addition, ECL biosensor is also used for the survey of the excretion physical examination in serum.
Description
Technical field
This disclosure relates to a kind of two-dimension nano materials-Ti3C2MXenes utilizes its reproducibility, and reduction gold chloride is formed
The compound of AuNPs-MXenes, AuNPs-MXenes compound dual catalytic Particles in Electrochemiluminescofce ofce Luminol and utilize alginic acid
The method of (SA) multidigit point effect building electrochemical luminescence sensor detection excretion body of sodium.
Background technique
Here statement only provides background information related with the disclosure, without necessarily constituting the prior art.
Excretion body be a kind of diameter in 30~100nm, with the vesica of double membrane structure, exocytosis can be passed through by various kinds of cell
Mode is secreted into microenvironment.Excretion body is present in most people body fluid, including blood, urine, saliva and breast milk.They by
Most of mammalian cell secretions, carry the factor and promote cell-cell communication, including mRNA, carbohydrate, protein
(CD63, CD81CD9 and EpCAM) and DNA.It is reported that allochthon anti-tumor immune response, diagnosing tumor and other during
It plays a significant role, and is the promising biomarker of early-stage cancer diagnosis.Therefore, the Gao Ling surveyed for excretion physical examination
Sensitivity method is not only valuable to clinical diagnosis, but also provides and see about the basic biochemical process of growth and metastasis of tumours
Solution, while additionally aiding the related mechanism of further research anti-tumor immune response.
So far, the various methods surveyed for excretion physical examination, including Western blotting, flow cytometry have been developed
Or enzyme-linked immunosorbent.These methods have certain disadvantages, such as the behaviour of expensive instrument, complicated technical skills and time-consuming
Make etc..Therefore, developing simple, sensitive and reliable excretion body detecting method is a huge challenge.In recent years, electroluminescent chemistry
(ECL) is shone as a kind of powerful analytical technology, because due to its high sensitivity, rapidity, property and low cost easy to control.It
It is widely used in the detection of the substances such as protein, DNA, enzyme, therefore, these advantages based on ECL, it can be expected to be applied to outer
Physical examination survey is secreted, the activity of excretion body is analyzed.
Summary of the invention
For background technique, this disclosure relates to a kind of dual catalytic based on two titanium carbide-MXenes and gold nano grain
Particles in Electrochemiluminescofce ofce Luminol biosensor and application.
Specifically, this disclosure relates to the technical solution of following aspect:
In first typical embodiment of the disclosure, a kind of two-dimentional transition metal carbide-nucleic acid adaptation is provided
Body probe, its main feature is that: the probe includes nanometer sheet Ti3C2MXenes, amino acid and be modified with carboxyl CD63 protein nucleic acid it is suitable
Ligand, the nanometer sheet Ti3C2MXenes is connected by Ti-N key with amino acid, and amino acid passes through amido bond and aptamer
It is connected.
In second typical embodiment of the disclosure, the two-dimentional transition metal carbide-nucleic acid adaptation is provided
The preparation method of body probe, this method comprises: by nanometer sheet Ti3C2MXenes and amino acid are placed in water after mixing, are stirred
It mixes, isolated sediment, obtained sediment is reacted with aptamers progress amide can be obtained two-dimentional transition metal carbonization
Object-nucleic acid aptamer probe.
In the typical embodiment of third of the disclosure, a kind of bio-sensing being used cooperatively with the probe is provided
Device electrode, comprising: surface modification has the base electrode, sodium alginate and the CD63 protein nucleic acid for being modified with amino of polyacrylamide
Aptamers；
The surface modification have polyacrylamide base electrode and be modified with amino CD63 protein nucleic acid aptamers it is equal
Pass through the connection of carboxylic ammonia reaction and sodium alginate.
In the 4th typical embodiment of the disclosure, the preparation method of the biological sensor electrode is provided, it should
Method includes: to drip to polyacrylamide solution on base electrode, dry；Then base electrode is immersed in 1- (3- diformazan ammonia
Base propyl) in -3- ethyl-carbodiimide hydrochloride (EDC), n-hydroxysuccinimide (NHS) and the mixed solution of sodium alginate
Hatched；Then base electrode is entered in aptamer solution and is hatched, biological sensor electrode is obtained.
In the 5th typical embodiment of the disclosure, a kind of electrogenerated chemiluminescence (ECL) biosensor is provided,
The biosensor includes the two-dimentional transition metal carbide-nucleic acid aptamer probe and the biological sensor electrode, when
In the presence of excretion body, the two dimension transition metal carbide-nucleic acid aptamer probe, the biological sensor electrode and excretion body
It is formed sandwich structure (two-dimentional transition metal carbide-nucleic acid aptamer probe-excretion body-biological sensor electrode), and will
The sandwich structure is immersed in chlorauric acid solution, and AuNPs- two dimension transition metal carbide-aptamer is formed in situ and visits
Needle-excretion body-biological sensor electrode, as electrogenerated chemiluminescence (ECL) biosensor.
In the 6th typical embodiment of the disclosure, a kind of kit of electrogenerated chemiluminescence is provided, the reagent
Box includes the two-dimentional transition metal carbide-nucleic acid aptamer probe, the biological sensor electrode, luminol and gold chloride
Solution.
In the 7th typical embodiment of the disclosure, the two-dimentional transition metal carbide-nucleic acid adaptation is provided
Body probe, the biological sensor electrode or the electrogenerated chemiluminescence (ECL) biosensor or the kit are being adopted
With the application in electroluminescent chemiluminescence method detection excretion body.
In the 8th typical embodiment of the disclosure, a kind of side of the detection excretion body of non-diagnostic purpose is provided
Method, this method include using the two-dimentional transition metal carbide-nucleic acid aptamer probe, the biological sensor electrode or institute
Electrogenerated chemiluminescence (ECL) biosensor or the kit stated are using electrogenerated chemiluminescence method detection excretion body
Step.
Compared with the relevant technologies that the present inventor knows, the one of technical solution of the disclosure has following beneficial to effect
Fruit:
The application combination multidigit point recognition strategy and New Two Dimensional material-Ti3C2The reproducibility of MXenes forms AuNPs-
MXenes-Apt designs highly sensitive electrochemical luminescence (ECL) sensing platform as ECL signal probe to detect Hela excretion
Body.In the strategy, sodium alginate (SA) polymer molecule can provide multidigit point identification interface with capture more aptamers with
The capture rate of excretion body is improved, this facilitates the sensitivity of improvement biosensor and AuNP-MXenes-Apt can be double
Catalysis luminol makes ECL signal enhancing again.Based on both dual amplification strategies, highly sensitive and highly selective life is obtained
Object sensor detects excretion body, in addition, ECL biosensor is also used for excretion physical examination in serum survey.The sensor can be made
For the feasibility tool for detecting excretion body, strong evidence is provided for the clinical diagnosis etc. of excretion body.
Detailed description of the invention
The Figure of description for constituting disclosure a part is used to provide further understanding of the disclosure, the signal of the disclosure
Property embodiment and its explanation for explaining the disclosure, do not constitute the improper restriction to the disclosure.
Fig. 1 is assembling and the mechanism schematic diagram of disclosure electrochemical luminescence sensor.
Fig. 2 is Ti3AlC2SEM figure.
Fig. 3 is the TEM figure of MXenes.
Fig. 4 is the XRD diagram of MXenes removing front and back.
Fig. 5 is the signal response of the electrochemical luminescence sensor of various concentration excretion body；A-h is represented: 102,5×102,
103,2.5×103,5×103,104, 5 × 104,105particles/μL。
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the disclosure.Unless another
It indicates, all technical and scientific terms used herein has usual with disclosure person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the disclosure.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation and/or their combination.
As background technique is introduced, a kind of highly sensitive and highly selective biosensor is needed to detect excretion
Body, in view of this, it is suitable to provide a kind of two-dimentional transition metal carbide-nucleic acid in first typical embodiment of the disclosure
Ligand probe, its main feature is that: the probe includes nanometer sheet Ti3C2MXenes, amino acid and the CD63 protein nucleic acid for being modified with carboxyl
Aptamers, the nanometer sheet Ti3C2MXenes is connected by Ti-N key with amino acid, and amino acid is adapted to by amido bond with nucleic acid
Body is connected.
What disclosure amplifying nucleic acid aptamers were specifically bound is the CD63 albumen in excretion body surface face.At one of the disclosure or
In some embodiments, the aptamer of the CD63 albumen includes but is not limited only to following sequence: 5'--
TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA can also include as shown in SEQ ID NO.1
The aptamer of other specific binding CD63 albumen.
The design of the probe can retain the advantageous property of MXenes itself: big specific surface area, good conductivity, high urges
Change property and superpower reproducibility, moreover it is possible to make the surface MXenes with amino, on the electrode convenient for modification.This is to utilize ammonia
Interaction force between base acid and MXenes combines, and forms Ti-N key, makes MXenes surface modification amino.With before study
It is compared by electrostatic adsorption, it is more more stable than electrostatic adsorption in the way of current this modification, it can make
The surface MXenes has amino, carries out the modification of next step.
In one of the disclosure or some specific embodiments, the amino acid is glycine or leucine, is passed through
Verification experimental verification, glycine or leucine can be with Ti3C2MXenes is preferably combined.
In second typical embodiment of the disclosure, the two-dimentional transition metal carbide-nucleic acid adaptation is provided
The preparation method of body probe, this method comprises: by nanometer sheet Ti3C2MXenes and amino acid are placed in water after mixing, are stirred
It mixes, isolated sediment, obtained sediment is reacted with aptamers progress amide can be obtained two-dimentional transition metal carbonization
Object-nucleic acid aptamer probe.
Further, the Ti3C2Ingredient proportion (0.5~1) mg:(4~6 of MXenes, amino acid and water) mg:(10~
40)mL；18~36h is stirred at room temperature.
Further, the temperature of the amide reaction is 35~40 DEG C, and the time is 0.5~1.5h.
Ti3C2MXenes have good electron transfer capacity, excellent catalytic capability, good biocompatibility and
Superpower reproducibility.In addition, Ti3C2MXenes big specific surface area also becomes the good carrier for loading more biomolecule,
To significantly improve the sensitivity of biosensor.Further, the nanometer sheet Ti3C2The preparation method of MXenes, including with
Lower step: LiF is added in the hydrochloric acid that concentration is 8~10mol/L, stirring, then by Ti3AlC2Powder is added in this mixture
And it is stirred 18~36 hours at 30~40 DEG C；Then by solution centrifuge washing, make pH >=6, discard supernatant liquid, adds in precipitating
Entering water, be centrifuged, discards supernatant liquid, water is added in continuation in precipitating, under the protection of nitrogen, 0.5~1.5h of ultrasound, finally, will
Solution centrifugation, retains supernatant, as nanometer Ti3C2MXenes dispersion liquid.
In the typical embodiment of third of the disclosure, a kind of bio-sensing being used cooperatively with the probe is provided
Device electrode, comprising: surface modification has the base electrode, sodium alginate and the CD63 protein nucleic acid for being modified with amino of polyacrylamide
Aptamers；
The surface modification has the base electrode of polyacrylamide and aptamer to pass through carboxylic ammonia to react and alginic acid
The connection of sodium.
Further, described matrix electrode is glass-carbon electrode.
Sodium alginate (C6H7O6Na) n is mainly made of the sodium salt of alginic acid, by beta-D-mannuronic acid (M unit) and α-
The copolymerization that L- guluronic acid (G unit) relies on β -1,4- glucosides key connection and is made of GM, MM and GG segment of different proportion
Object, surface carboxyl containing there are many, can provide more recognition sites, and its good hydrophilic property, and adhesive force is low, have excellent performance,
And have biocompatibility, therefore the disclosure using its excellent property can it is significant improve biosensor sensitivity and
Selectivity.In polydispersity molecules group, usual Mw > Mn.The coefficient of Mw/Mn is monodispersity index, the finger of sodium alginate commodity
Number classics range is 1.5~2.5.
In the 4th typical embodiment of the disclosure, the preparation method of the biological sensor electrode is provided, it should
Method includes: to drip to polyacrylamide solution on base electrode, is hatched to dry；Then base electrode is immersed in 1- (3- bis-
Methylaminopropyl) -3- ethyl-carbodiimide hydrochloride (EDC), n-hydroxysuccinimide (NHS) and sodium alginate mixing it is molten
Hatched in liquid；Then base electrode is entered in aptamer solution and is hatched, biological sensor electrode is obtained.
Further, 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC), N- hydroxysuccinimidyl
The volume ratio of acid imide (NHS) and sodium alginate is 0.8~1.2:0.8~1.2:1.8~2.2, the concentration of EDC is 80~
120 μM, the concentration of NHS is 300~500 μM, and the concentration of sodium alginate is 0.5~1.5mg/ml；35~40 DEG C hatch 1~
3h。
Further, then base electrode is entered in aptamer solution and is hatched, incubation condition are as follows: 35~40
DEG C hatching 1~3h.
Further, the weight average molecular weight of the polyacrylamide are as follows: MW >=3,000,000.
In the 5th typical embodiment of the disclosure, a kind of electrogenerated chemiluminescence (ECL) biosensor is provided,
The biosensor includes the two-dimentional transition metal carbide-nucleic acid aptamer probe and the biological sensor electrode, when
In the presence of excretion body, the two dimension transition metal carbide-nucleic acid aptamer probe, the biological sensor electrode and excretion body
It is formed sandwich structure (two-dimentional transition metal carbide-nucleic acid aptamer probe-excretion body-biological sensor electrode), and will
The sandwich structure is immersed in chlorauric acid solution, and AuNPs- two dimension transition metal carbide-aptamer is formed in situ and visits
Needle-excretion body-biological sensor electrode, as electrogenerated chemiluminescence (ECL) biosensor.
In the 6th typical embodiment of the disclosure, a kind of kit of electrogenerated chemiluminescence is provided, the reagent
Box includes at least the two-dimentional transition metal carbide-nucleic acid aptamer probe, the biological sensor electrode, chlorauric acid solution
And luminol.
In the 7th typical embodiment of the disclosure, the two-dimentional transition metal carbide-nucleic acid adaptation is provided
Body probe, the biological sensor electrode or the electrogenerated chemiluminescence (ECL) biosensor or the kit are being adopted
With the application in electroluminescent chemiluminescence method detection excretion body.
In the 8th typical embodiment of the disclosure, a kind of side of the detection excretion body of non-diagnostic purpose is provided
Method, this method include using the two-dimentional transition metal carbide-nucleic acid aptamer probe, the biological sensor electrode or institute
Electrogenerated chemiluminescence (ECL) biosensor or the kit stated are using electrogenerated chemiluminescence method detection excretion body
Step.
It include: that the biological sensor electrode is dipped to excretion body to be measured using the method that the kit is detected
In solution, makes the attachment of excretion body on a biosensor electrode, be then dipped to the biological sensor electrode for adhering to excretion body
In two-dimentional transition metal carbide-nucleic acid aptamer probe solution, it is attached to probe on the excretion body of biological sensor electrode,
The biosensor of excretion body is carried to form probe and biological sensor electrode folder, then the biosensor is immersed in chlorine
In auric acid solution, make nanogold particle reduction on the surface of electrode composite, and then be prepared into surface and be attached with nanogold
Probe and the biological sensor electrode folder of grain carry the electrochemical luminescence biosensor of excretion body, pass to the electrochemical luminescence biology
Sensor carries out electrochemical luminescence detection in luminol solution.
Specific method includes:
(1) preparation of standard solution: the excretion body standard solution of one group of various concentration is prepared；
(2) biological sensor electrode drafting of working curve: is dipped to the excretion body standard of various concentration respectively
In solution, makes the attachment of excretion body on a biosensor electrode, be then dipped to the biological sensor electrode for adhering to excretion body
In two-dimentional transition metal carbide-nucleic acid aptamer probe solution, it is attached to probe on the excretion body of biological sensor electrode,
The biosensor of excretion body is carried to form probe and biological sensor electrode folder, then the biosensor is immersed in chlorine
In auric acid solution, make nanogold particle reduction on the surface of electrode composite, and then be prepared into surface and be attached with nanogold
Probe and the biological sensor electrode folder of grain carry the electrochemical luminescence biosensor of excretion body, in the case where photomultiplier tube is 600V
Electrochemical luminescence detection is carried out in luminol solution to the electrochemical luminescence biosensor, obtains the excretion body of various concentration
It is bent to draw linear relationship for the ECL intensity of standard solution, concentration and corresponding ECL intensity further according to excretion body standard solution
Line；
(3) detection of sample: the ECL intensity of excretion liquid solution to be measured is measured according to the method in step (2), further according to institute
Linear relationship curve is stated, the concentration of excretion body in excretion liquid solution to be measured is obtained.
The detection method is used for the detection excretion body of non-diagnostic purpose, can study the related mechanism of anti-tumor immune response
Deng.
In the disclosure, Ti is found3C2MXenes can be used as catalyst and reducing agent, utilize its reproducibility, reduction
HAuCl4AuNPs-MXenes compound is formed, for improving the ECL of luminol.To which we have developed a kind of based on the more of SA
High-sensitivity biological sensor platform of the site identification in conjunction with AuNPs-MXenes catalysis Particles in Electrochemiluminescofce ofce Luminol is for outer
Secrete physical examination survey.Detection is limited to 30/microlitre, and the conventional method and applicant that detection limit is relatively higher than enzyme linked immunological are ground early period
The ECL biosensor studied carefully.In addition, being based on dual amplification strategy, the detection of other excretion bodies all achieves success, this table
The bright platform is feasible.Therefore, the multidigit point identification based on SA and AuNPs-MXenes-Aptamer nano-probe combine
Biosensor provides strong tool for assessment exosome surface protein expression, and opens and be metabolized to allochthon
The neodoxy of physiological function and clinical diagnosis in journey and drug screening.
The assembling of electrochemical luminescence sensor and experimental principle:
Assembling and the mechanism of electrochemical luminescence sensor are as shown in Figure 1, base electrode glass-carbon electrode (GCE)] surface modification
Polyacrylamide (PAM), to be reacted according to carboxylic ammonia, can have since amino is contained on the surface PAM in its surface-modifying surface
The sodium alginate (SA) of many carboxyls, can increase the active site of aptamers combination in this way, and more aptamers modifications is made to exist
On electrode, to capture more excretion bodies.Then synthesis MXenes-Aptamer (MXenes-glycine-Apt) is as spy
Needle forms sandwich structure in conjunction with excretion body, and reproducibility finally superpower using MXenes impregnates the electrode being completed
In gold chloride (HAuCl4) in, AuNPs-MXenes-Aptmer compound, the AuNPs-of formation are formed in electrode surface
The electrochemical luminescence of MXenes-Aptmer dual catalytic luminol, is reflected using the power of the electrochemical luminescence signals of generation
Capture excretion body number, thus achieve the purpose that detect excretion body.Electrochemical luminescence signals are relatively strong then in electrode surface
AuNPs-MXenes-Aptmer is more, thus indirectly illustrate that excretion body on the electrode is more, it can be according to electrochemical signals
Power come reflect this sensor capture excretion body number.
In order to enable those skilled in the art can clearly understand the technical solution of the disclosure, below with reference to tool
The technical solution of the disclosure is described in detail in the embodiment of body.
Experiment reagent and material
Aptamers are 5'-COOH-TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA, 5'-
NH2- TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA is obtained from the raw work biotechnology in Shanghai
Services Co., Ltd.Ti3AlC2(98%) purchased from the graceful scientific Co., Ltd (BeiJing, China) of Fox.Polyacrylamide (PAM) and Shandong
Minot is purchased from Sigma-Aldrich.HAuCl4·3H2O (48%, w/w) is obtained from Shanghai Reagent (Chinese Shanghai).Sea
Mosanom (SA) is purchased from Aladdin (China) product number S100128,1- (3- (dimethylamino) propyl) -3- ethyl carbonization two
Inferior amine salt hydrochlorate (EDC) and n-hydroxysuccinimide sodium salt (NHS), ethylenediamine (EDA) are purchased from Beijing Chemical Co., Ltd.
(BeiJing, China), lithium fluoride are purchased from Shanghai Aladdin Bioisystech Co., Ltd, other analytical grade reagents are all from traditional Chinese medicines chemistry
Reagent Company (China).
Laboratory apparatus
Electrochemical workstation, electrochemical luminescence work station, using three-electrode system (reference electrode: Ag/AgCl electrode, it is right
Electrode: platinum electrode, working electrode: glass-carbon electrode), scanning electron microscope SEM, transmission electron microscope, ultraviolet spectrometry light
Spend instrument, Fourier infrared spectrograph etc..
Embodiment 1
1, the synthesis of MXenes-glycine-Aptmer nano-probe
10ml is added in 0.8g LiF, in the hydrochloric acid of 9mol/L, is stirred 5 minutes, then by Ti3AlC2(0.5g) powder adds
Enter in this mixture and is stirred 24 hours at 35 DEG C.Then by solution centrifuge washing 7-8 times, makes pH >=6, discards supernatant liquid,
40-50mL deionized water is added in precipitating, with 3500rpm centrifugation 60 minutes, discards supernatant liquid, continuation is added in precipitating
40-50mL deionized water, under the protection of nitrogen, ultrasonic 1h, finally, solution is retained supernatant with 3500rpm centrifugation 60 minutes
Liquid (as nanometer Ti3C2MXenes dispersion liquid), and saved backup at 4 DEG C.
The glycine (glycine) of 5mg is dissolved in 20ml deionized water and is stirred at room temperature.Then, it is added dropwise
10mL concentration is the Ti of 0.075mg/ml3C2MXenes dispersion liquid is simultaneously stirred at room temperature 24 hours, then with 12000r centrifugation 20
Minute, liquid is discarded supernatant, precipitating is labeled as MXenes-glycine, spare.
EDC (400mM) and NHS (100mM) and Aptamer (1uM, 5'-COOH-TTTTTT CAC CCC ACC TCG
CTC CCG TGA CAC TAA TGC TA) mixture activates 1 hour at 37 DEG C.Hereafter, 200 μ L are obtained
Ti3C2MXenes-glycine is added in Aptmer solution at 37 DEG C, 1h is reacted, finally, mixture is centrifuged with 12000rpm
10 minutes, liquid is discarded supernatant, wash and deionized water is added.
2, the covalent reaction of the assembling of electrode and peptide chain on the electrode
(1) glassy carbon electrode surface pre-processes
By glass-carbon electrode (GCE) with 0.3 μm of Al2O3Powder carries out sanding and polishing processing on chamois leather, then uses second respectively
Alcohol, water are cleaned by ultrasonic 3min, are dried up electrode surface with pure nitrogen.
The glass-carbon electrode of cleaning drying makees working electrode, and Ag/AgCl makees reference electrode, and platinum filament is made to electrode, the potassium ferricyanide
In solution, -0.2~0.6V, 100mV/s, scanning CV to stabilization.Repeatedly, until the redox potential of glass-carbon electrode is poor
In 80mV or so, glass-carbon electrode is eluted with water, is dried with nitrogen.
(2) assembling of electrochemical luminescence biosensor
GCE after PAM moditied processing: taking concentration is that the 6 μ l of PAM of 0.1mg/ml drips to glassy carbon electrode surface, is incubated at 37 DEG C
Change to dry, electrode is then immersed in 100 μM of EDC, in the SA of 400 μM of NHS and 1mg/ml (volume ratio is according to 1:1:
2), hatch 2h at 37 DEG C.Electrode is then immersed in 1 μM of (40 μ L) Aptmer (5'-NH2-TTTTTTCAC CCC ACC TCG
CTC CCG TGA CAC TAA TGC TA) in, hatch 2h at 37 DEG C, obtains Aptmer/SA/PAM/GCE after cleaning drying.
Aptmer/SA/PAM/GCE is immersed in the Hela excretion body of various concentration, the 2h in 37 DEG C of environment.It cleans
Exosomes/Aptmer/SA/PAM/GCE is obtained after drying.
After the electrode for having captured excretion body is cleaned drying with distilled water, it is placed in probe solution and hatches 2h at 37 DEG C, to
It is cleaned, is dried with nitrogen, then by assembled electrode (MXenes-Aptmer/ with distilled water after fully reacting
Exosomes/Aptmer/SA/PAM/GCE) it is immersed in the HAuCl of 2mg/ml41h in solution, then dries up electrode washing, i.e.,
The electrochemical luminescence biosensor prepared.
Due to CD63 albumen be on excretion body it is generally existing, only content is different, therefore using the sensor
Platform monitors the excretion body of different cell secretions.The application using this method also have detected liver cancer cells (HepG2) excretion body and
Ovarian cancer cell (OVCAR) excretion etc..
3, the discussion of experimental result
Fig. 2 is the Ti before removing3AlC2Scanning electron microscope schematic diagram.
The TEM figure that Fig. 3 is the MXenes after removing.As seen from the figure by removing.Form the MXenes of nanometer sheet.
Fig. 4 is the XRD diagram of removing front and back MXenes.
Fig. 5 is the signal response of the electrochemical luminescence sensor after the catalysis of various concentration excretion body, is sent out using this electrochemistry
Optical biosensor analyzes excretion body.As seen from Figure 5, with the increase of excretion bulk concentration, electrochemical luminescence signals by
It is cumulative big.It is 10 in the concentration of excretion body2-5*105It is a/microlitre within the scope of, the sizes of electrochemical luminescence signals is dense with excretion body
The logarithm of degree is in a linear relationship.
Embodiment 2
1, the synthesis of MXenes-glycine-Aptmer nano-probe
With embodiment 1.
2, the assembling of electrode and the assembling of sensor
(1) glassy carbon electrode surface pre-processes
With embodiment 1
(2) assembling of electrochemical luminescence biosensor
GCE after PAM moditied processing: taking concentration is that the 6 μ l of PAM of 0.1mg/ml drips to glassy carbon electrode surface, is incubated at 37 DEG C
Change to dry, electrode is then immersed in 100 μM of EDC, in the SA of 400 μM of NHS and 1mg/ml (volume ratio is according to 1:1:
2), hatch 2h at 37 DEG C.Then electrode is immersed in 0.8 μM of (40 μ L) Aptmer, hatches 2h at 37 DEG C, is obtained after cleaning drying
To Aptmer/SA/PAM/GCE.Aptmer/SA/PAM/GCE is immersed in the excretion body of various concentration, in 25 DEG C of environment
Middle 1h.Exosomes/Aptmer/SA/PAM/GCE is obtained after cleaning drying.
After the electrode for having captured excretion body is cleaned drying with distilled water, it is placed in probe solution and hatches 2h at 37 DEG C, to
It is cleaned after fully reacting with distilled water, is dried with nitrogen, then assembled electrode is immersed in the HAuCl of 2mg/ml4
1h in solution forms AuNPs-MXenes--Aptmer/exosomes/Aptmer/SA/PAM/GCE, then blows electrode washing
It is dry to get to the electrochemical luminescence biosensor prepared.
Embodiment 3
1, the synthesis of MXenes-glycine-Aptmer nano-probe
With embodiment 1
2, the assembling of electrode and the assembling of sensor
(1) glassy carbon electrode surface pre-processes
With embodiment 1.
(2) assembling of electrochemical luminescence biosensor
GCE after PAM moditied processing: taking concentration is that the 6 μ l of PAM of 0.1mg/ml drips to glassy carbon electrode surface, is incubated at 37 DEG C
Change to dry, electrode is then immersed in 100 μM of EDC, in the SA of 400 μM of NHS and 1mg/ml (volume ratio is according to 1:1:
2), hatch 2h at 37 DEG C.Then electrode is immersed in 1.2 μM of (40 μ L) Aptmer, hatches 2h at 37 DEG C, is obtained after cleaning drying
In the excretion body that Aptmer/SA/PAM/GCE is immersed in various concentration to Aptmer/SA/PAM/GCE., in 50 DEG C of environment
In 30 minutes.Exosomes/Aptmer/SA/PAM/GCE is obtained after cleaning drying.
After the electrodes for having captured excretion body are cleaned drying with distilled water, it is placed in probe solution and hatches at 37 DEG C
1h is dried with nitrogen to be cleaned after fully reacting with distilled water, assembled electrode is then immersed in 2mg/ml's
HAuCl41h in solution forms AuNPs-MXenes-Aptmer/exosomes/Aptmer/SA/PAM/GCE, then by electrode
Drying is rinsed to get to the electrochemical luminescence biosensor prepared.
Above-described embodiment is the preferable embodiment of the disclosure, but embodiment of the present disclosure is not by above-described embodiment
It limits, made changes, modifications, substitutions, combinations, simplifications under other any spiritual essence and principles without departing from the disclosure,
It should be equivalent substitute mode, be included within the protection scope of the disclosure.
SEQUENCE LISTING
<110>University Of Qingdao
<120>a kind of to be passed based on the dual catalytic Particles in Electrochemiluminescofce ofce Luminol biology of gold nano grain and two titanium carbide MXenes
Sensor
<130> 2018
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 38
<212> DNA
<213>aptamer
<400> 1
ttttttcacc ccacctcgct cccgtgacac taatgcta 38
Claims (10)
1. a kind of two dimension transition metal carbide-nucleic acid aptamer probe, it is characterized in that: the probe includes nanometer sheet Ti3C2
MXenes, amino acid and the CD63 protein nucleic acid aptamers for being modified with carboxyl, the nanometer sheet Ti3C2MXenes passes through Ti-N key
It is connected with amino acid, amino acid is connected by amido bond with aptamer.
2. two dimension transition metal carbide-nucleic acid aptamer probe as described in claim 1, it is characterized in that: the nucleic acid is suitable
The sequence of ligand are as follows: 5'-COOH-TTTTTTCAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA.
3. the preparation method of two dimension transition metal carbide-nucleic acid aptamer probe of any of claims 1 or 2, characterized in that
Method includes the following steps: by nanometer sheet Ti3C2MXenes and amino acid are placed in water after mixing, and stirring separates
To sediment, obtained sediment and aptamers carried out amide react two-dimentional transition metal carbide-nucleic acid can be obtained to fit
Ligand probe；
Further, the amino acid is glycine or leucine；
Further, the Ti3C2The ingredient proportion of MXenes, amino acid and water are (0.5~1) mg:(4~6) mg:(10~
40)mL；18~36h is stirred at room temperature；
Further, the temperature of the amide reaction is 35~40 DEG C, and the time is 0.5~1.5h.
4. a kind of biological sensor electrode being used cooperatively with probe of any of claims 1 or 2, characterized in that include: surface
It is modified with the base electrode, sodium alginate and the CD63 protein nucleic acid aptamers for being modified with amino of polyacrylamide；
The surface modification has the base electrode of polyacrylamide to pass through with the CD63 protein nucleic acid aptamers for being modified with amino
The connection of carboxylic ammonia reaction and sodium alginate；
Further, described matrix electrode is glass-carbon electrode；
Further, the sequence of the aptamer are as follows: 5'-NH2-TTTTTTCAC CCC ACC TCG CTC CCG TGA
CAC TAA TGC TA。
5. the preparation method of biological sensor electrode as claimed in claim 4, characterized in that this method comprises: by polyacrylamide
Amine aqueous solution drips on base electrode, hatches to dry；Then base electrode is immersed in 1- (3- dimethylamino-propyl) -3- ethyl carbon
Hatched in diimmonium salt hydrochlorate (EDC), n-hydroxysuccinimide (NHS) and the mixed solution of sodium alginate；Then will
Base electrode, which enters in aptamer solution, is hatched, and biological sensor electrode is obtained.
6. preparation method as claimed in claim 5, it is characterized in that: the 1- (3- dimethylamino-propyl) -3- ethyl carbon two is sub-
The volume ratio of amine hydrochlorate (EDC), n-hydroxysuccinimide (NHS) and sodium alginate is 0.8~1.2:0.8~1.2:
The concentration of 1.8~2.2, EDC are 80~120 μM, and the concentration of NHS is 300~500 μM, the concentration of sodium alginate is 0.5~
1.5mg/ml；In 35~40 DEG C of 1~3h of hatching；
Further, then base electrode is entered in aptamer solution and is hatched, incubation condition are as follows: 35~40 DEG C incubate
Change 1~3h.
7. a kind of electrogenerated chemiluminescence (ECL) biosensor, it is characterized in that: the biosensor includes claims 1 or 2 institute
Two-dimentional transition metal carbide-the nucleic acid aptamer probe stated and biological sensor electrode as claimed in claim 4, when excretion body
In the presence of, the two dimension transition metal carbide-nucleic acid aptamer probe, the biological sensor electrode and excretion body form three
Mingzhi's structure, and the sandwich structure is immersed in chlorauric acid solution, AuNPs- two dimension transition metal carbide-is formed in situ
Nucleic acid aptamer probe-excretion body-biological sensor electrode, as electrogenerated chemiluminescence (ECL) biosensor.
8. a kind of kit of electrogenerated chemiluminescence, it is characterized in that: the kit includes at least of any of claims 1 or 2 two
Tie up transition metal carbide-nucleic acid aptamer probe, biological sensor electrode as claimed in claim 4, chlorauric acid solution and Shandong
Minot.
9. two dimension transition metal carbide-nucleic acid aptamer probe, biology as claimed in claim 4 of any of claims 1 or 2
Sensor electrode or electrogenerated chemiluminescence as claimed in claim 7 (ECL) biosensor or kit according to any one of claims 8
Using the application in electrogenerated chemiluminescence method detection excretion body.
10. a kind of method of the detection excretion body of non-diagnostic purpose, characterized in that this method comprises:
Biological sensor electrode as claimed in claim 4 is dipped in excretion liquid solution to be measured, excretion body is made to be attached to biology
On sensor electrode, the biological sensor electrode for adhering to excretion body is then dipped to two-dimentional transition of any of claims 1 or 2
In metal carbides-nucleic acid aptamer probe solution, it is attached to probe on the excretion body of biological sensor electrode, to form
Probe and biological sensor electrode folder carry the biosensor of excretion body, then the biosensor is immersed in chlorauric acid solution
In, make nanogold particle reduction on the surface of electrode composite, and then be prepared into the probe that surface is attached with nanogold particle
The electrochemical luminescence biosensor for carrying excretion body is pressed from both sides with biological sensor electrode, to the electrochemical luminescence biosensor in Shandong
Electrochemical luminescence detection is carried out in minot solution.
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| PCT/CN2019/112594 WO2020114124A1 (en) | 2018-12-03 | 2019-10-22 | Electrochemiluminescence biosensor based on luminol double catalyzed by aunps and mxenes |
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