CN110057877A - The biosensor and its preparation method for being used to detect tumour cell of repeatable modification - Google Patents
The biosensor and its preparation method for being used to detect tumour cell of repeatable modification Download PDFInfo
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Abstract
The present invention is a kind of repeatable modification for detecting the biosensor and its preparation method of tumour cell.Tetrahedron-aptamers lead to Au-S key and independently fill and is fixed on gold electrode, thus efficiently, specifically capture human breast cancer cell.The PCN-224-Pt/ horseradish peroxidase of synthesis/bis- aptamers/ferrohemes/G- tetrad novel metal organic frame nano-probe is introduced on cell sensing interface by the combination of aptamers and cell then, crosses hydroquinone (HQ)-hydrogen peroxide (H by three kinds of horseradish peroxidase, nano enzyme (Pt nano particle) and DNA enzymatic (ferroheme/G- tetrad) enzymatics2O2).The present invention can be used for the detection of tumour cell, have many advantages, such as highly selective, highly sensitive and simplicity quickly, electrode can modify repeatedly.
Description
Technical field
The present invention relates to electrochemica biological sensors and preparation method thereof, more particularly to based on novel metal organic frame
Double aptamers 3-D nano, structures, repeatable modification electro-chemical cells sensor is connected with tetrahedron.Belong to biomedicine
Technical field.
Background technique
Cancer is that the world today threatens human life's most common disease safely.It is well known that the early diagnosis of cancer can
Effectively improve the cure rate of cancer patient.It accurately delicately identifies, detect early diagnosis and clinic of the tumour cell for cancer
Treatment has great importance.Currently, the conventional method for tumour cell detection includes immunohistochemistry, fluidic cell point
Analysis method and Reverse transcription-poly-merase chain reaction etc..Although these methods popularity rate with higher, the big multioperation of these methods
It is cumbersome, experimental cost is high, needs professional operator and advanced instrument and equipment etc..Up to the present, it more induces one to close
What is infused is the super sensitivity detection that electrochemical measuring technique realizes tumour cell.Electrochemica biological sensor has easy to operate, clever
It the advantages that sensitivity height and rapid detection, is very suitable to for carrying out tumour cell detection.
Summary of the invention
One of the objects of the present invention is to provide a kind of connect double aptamers with tetrahedron based on metal organic frame can
The electro-chemical cells sensor modified repeatedly.The second object of the present invention is the preparation method and application of the sensor in tumour
The detection of cell.
In order to achieve the goal above, the biosensor for being used to detect tumour cell of repeatable modification of the invention, with
Gold electrode is as detection interface, and tetrahedron connects double aptamers and is incorporated in gold electrode surfaces by golden sulfide linkage, and PCN-224-Pt/ is peppery
Root peroxidase/bis- aptamers/ferrohemes/G- tetrad novel metal organic frame nano-probe is captured thin by aptamers
Born of the same parents and be connected to cell sensing interface.
Repeatable modification of the invention for detect tumour cell biosensor the preparation method comprises the following steps: tetrahedron-
Aptamers lead to Au-S key and independently fill and is fixed on gold electrode, thus efficiently, specifically capture human breast cancer cell.It then will synthesis
PCN-224-Pt/ horseradish peroxidase/bis- aptamers/ferrohemes/G- tetrad novel metal organic frame nano-probe
It is introduced on cell sensing interface by the combination of aptamers and cell, passes through horseradish peroxidase, nano enzyme (Pt nanometers
Grain) and three kinds of enzymatics of DNA enzymatic (ferroheme/G- tetrad) cross hydroquinone (HQ)-hydrogen peroxide (H2O2), it further realizes
The amplification of electrochemical signals.After experiment, applied voltage destroys Au-S key and regenerates naked gold electrode surfaces, can be used for
Again, modify repeatedly.Specific following steps:
(1) pre-treatment of gold electrode: gold electrode uses Piranha washing lotion to impregnate 15min first, and it is enterprising to be subsequently placed in polishing cloth
Row polishing, it is sulfuric acid activated until signal stabilization after ultrasonic treatment, then with 0.5M.
(2) fixation of the aptamers of tetrahedron connection: the aptamers AS1411- of 10 μM of the single-stranded connection of tetrahedron is taken respectively
A, in single-stranded AS1411-B, AS1411-C and AS1411-D Yu Sizhi PCR pipe of tetrahedron, three chloroethenes of isometric 1mM are respectively added
Base phosphate reacts 1h.Above-mentioned solution is mixed in same PCR pipe, and 10min is reacted under the conditions of 95 DEG C, is cooled to 4 at once
10min is reacted under the conditions of DEG C, can obtain the aptamers AS1411 three-dimensional structure of tetrahedron connection.The aptamers MUC1 of tetrahedron connection
The three-dimensional structure same Fa Ke get of MUC1-A, MUC1-B, MUC1-C and MUC1-D.10 μ L, two kinds of adaptation liquid solutions are taken to be added drop-wise to preceding place
The gold electrode managed reacts 3h under the conditions of 37 DEG C.Two kinds of aptamers are independently filled by Au-S key to be fixed on gold electrode, can be high
Effect specifically captures human breast cancer cell line Bcap-37.
The sequence of the aptamers AS1411-A of the single-stranded connection of tetrahedron of the AS1411 three-dimensional structure are as follows: 5 '-ACA
TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ATT TTT
TTT TTG GTG GTG GTG GTT GTG GTG GTG GTG G-3’。
The sequence of the aptamers MUC1-A of the single-stranded connection of tetrahedron of the MUC1 three-dimensional structure are as follows: 5 '-ACA TTC
CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ATT TTT TTT
TTG CAG TTG ATC CTT TGG ATA CCC TGG-3’。
The sequence of the single-stranded B of tetrahedron of the AS1411 three-dimensional structure and MUC1 three-dimensional structure are as follows: 5 '-HS-TAT CAC
CAG GCA GTT GAC AGT GTA GCAAGC TGT AAT AGA TGC GAG GGT CCA ATA C-3’。
The sequence of the single-stranded C of tetrahedron of the AS1411 three-dimensional structure and MUC1 three-dimensional structure are as follows: 5 '-HS-TCA ACT
GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3’。
The sequence of the single-stranded D of tetrahedron of the AS1411 three-dimensional structure and MUC1 three-dimensional structure are as follows: 5 '-HS-TTC AGA
CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3’。
(3) fixation of multifunctional nano probe: then by the DNA enzymatic of synthesis (G- tetrad/ferroheme)-bis- aptamers-
PCN-224-Pt particle-horse-radish peroxidase nano probe is introduced into different cell concentrations (20,1 × 102, 1 × 103, 1 ×
104, 1 × 105, 1 × 106With 1 × 107Cells/mL on sensing interface), in 37 DEG C of reaction 1h.Then by electrode be placed in containing
3mM HQ and 1.5mM H2O2Phosphate buffered saline (PBS) (PBS) solution in, react 5min, then carry out differential pulse voltametry
Scanning.
The sequence of the AS1411 aptamers are as follows: 5 '-HS- (CH2)6-TTTGGGTAGGGCGGGTTGGG-TTTTTT-
GGTGGTGGTGGTTGTGGTGGTGGTGG-3’。
The sequence of the MUC1 aptamers are as follows: 5 '-HS- (CH2)6-TTTGGGTAGGGCGGGTTGGG-TTTTTT-
GCAGTTGATCCTTTGGATACCCTGG-3’。
(4) the repetition modification of gold electrode: after experiment, applied voltage -1.7~-0.9V destroys Au-S key and lays equal stress on new life
At naked gold electrode surfaces, can be used for modifying again, repeatedly.
Since this electro-chemical cells sensor has dual signal amplification, the super of breast cancer cell may be implemented
Sensitive Detection, 20~1 × 107There is good linear, selectivity and reproducibility, detection to be limited within the scope of cells/mL
6cells/mL, and Successful utilization is in the detection of blood sample.
The beneficial effects of the present invention are: the present invention constructs a kind of electro-chemical cells sensor of repeatable modification.The biography
Sensor realizes the detection to breast cancer cell using the strategy of dual signal amplification, has high sensitivity, and selectivity is good and easy
The advantages that quick.Method is freed with electrochemistry simultaneously and realizes that the repetition of gold electrode is modified, and is had in terms of diagnosis early period of cancer
Good response application prospect.
Detailed description of the invention
Fig. 1 is the schematic illustration of electro-chemical cells sensor.
Fig. 2 is the transmission electron microscope phenogram (A) of metal organic frame PCN-224 and metal organic frame PCN-224-Pt
The transmission electron microscope phenogram (B) of grain.
The electronic energy spectrum (A) and metal organic frame PCN-224-Pt particle that Fig. 3 is metal organic frame PCN-224
Electronic energy spectrum (B).
Fig. 4 is the phenogram of the agarose electrophoresis of tetrahedron and aptamers.
Fig. 5 is aptamers ratio optimization (A), cell incubation time-optimized (B) and spy incubation time optimize (C).
Fig. 6 be modified electrode reacted with cancer cell after DPV response diagram: a-g:20,1 × 102, 1 × 103, 1 × 104, 1 ×
105, 1 × 106and 1×107Cells/mL (A), the linearity curve (B) and difference of different cell concentrations and corresponding peak current are thin
The DPV response diagram (C) of born of the same parents.
Fig. 7 is the cell Trypan Blue figure (B) and exposed gold after electrochemical sensor is freed schematic diagram (A), freed
The impedance diagram (C) of electrode and the gold electrode regenerated.
In Fig. 1, (1) is AS1411 aptamers, and (2) are MUC1 aptamers, and (3) are horseradish peroxidase, and (4) are blood red
Element, (5) are gold electrode, and (6) are DNA tetrahedron-AS1411 aptamers, and (7) are DNA tetrahedron-MUC1 aptamers, and (8) are mercapto
Base hexanol, (9) are MCF-7 cell.
In Fig. 7, (10) are the cell collected, and (11) are microscope, and (12) are regenerated electrode, and (13) are impedance signal
Figure.
Specific embodiment
Below by specific embodiment, the present invention is further explained, and following embodiment facilitates those skilled in the art
The present invention is further understood, but is not intended to limit protection scope of the present invention.
Embodiment one: the preparation of cell sensor nano-probe
0.15g ZrOCl is added in round-bottomed flask2·8H2O, the third rouge of 0.05g phosphoric acid trichlorine (TCPP), 1.4g benzoic acid
With 50mL n,N-Dimethylformamide (DMF), 5h is stirred to react at 90 DEG C.Later, it is centrifuged 30min in 12000rpm, used
DMF is thoroughly washed three times.Product is stored in DMF solution, is protected from light.Then, by 1mL H2PtCl6(19.31mM) is added to PCN-
224 nano particles (2.5mg mL-1) in, 1h is stirred in 20mL water.Then, 2mL NaBH is added with vigorous stirring4(4mg
mL-1), it is stirred for 3h, obtains PCN-224-Pt nano particle.Finally mixture is centrifuged and is washed with water 3 times.Obtained PCN-
224-Pt particle is redispersed in water, and some of them are for verifying the accuracy and catalytic performance of compound, other are for closing
At hybridized nanometer probe.Using scanning electron microscope (SEM) and electronic energy spectrum (EDS) method to PCN-224-Pt nanometers of materials
The preparation of material is verified, and as a result sees Fig. 2 and Fig. 3.By observing H2O2Catalytic decomposition in PCN-224-Pt generates oxygen,
Determine the catalytic performance of PCN-224-Pt.
1mg PCN-224-Pt nanoparticle is redispersed in 9 buffer solution of 1mL PBS pH.Then add in the solution
Enter 50 μ L HRP (1mg mL-1), 25 μ L AS1411 aptamers (5 μM) and 25 μ L MUC1 aptamers (5 μM), stirred at 4 DEG C
Reaction is for 24 hours.Then, by ferroheme (0.1mg) at 4 DEG C with 7.4 buffer mixing 2h of PBS pH, after being centrifuged off supernatant,
It is cleaned with PBS buffer solution.Bis- aptamers/the ferrohemes of the PCN-224-Pt/HRP/ of preparation/G- tetrad hybridized nanometer probe, most
After be stored in PBS buffer solution, 4 DEG C preservation.
Embodiment two: tetrahedron-aptamers preparation
Aptamers AS1411-A, tetrahedron single-stranded AS1411-B, AS1411- of 10 μM of the single-stranded connection of tetrahedron are taken respectively
In C and AS1411-D Yu Sizhi PCR pipe, the trichloroethyl phosphate of isometric 1mM is respectively added, reacts 1h.Above-mentioned solution is mixed
Together in same PCR pipe, 10min is reacted under the conditions of 95 DEG C, reacts 10min under the conditions of being cooled to 4 DEG C at once, can obtain tetrahedron
The aptamers AS1411 three-dimensional structure of connection.Aptamers MUC1 three-dimensional structure MUC1-A, MUC1-B of tetrahedron connection,
The same Fa Ke get of MUC1-C and MUC1-D.It is characterized using tetrahedron of 3% Ago-Gel to preparation.At room temperature in 100V
Lower progress electrophoresis 75min.After separation, gel is imaged using fluorescence gel imaging system, as a result sees Fig. 4.
Embodiment three: the preparation of sensor
After gold electrode is impregnated 15min with Piranha washing lotion, it is rinsed with water completely, it is then mixed with 0.05 μm of aluminium oxide
Suspension is polished, and ultrasound, ethyl alcohol and each 3min of deionized water are then carried out.It is sulfuric acid activated until signal is steady with 0.5M again
Fixed, sulfuric acid activated range is -0.2 to 1.5V.Then certain density specific tetrahedron-adaptation liquid solution is added drop-wise to electrode
On, under the conditions of 4 DEG C, stand overnight.Electrode is then taken out, is rinsed with ultrapure water.Also ultrapure water is soaked in when storage
In, it is placed under the conditions of 4 DEG C.
Example IV: the condition optimizing of tumour cell detection
In view of multiple binding site of the MCF-7 cell on aptamers probe, MUC1 and AS141 aptamers can be made
For detection probe.In order to obtain higher sensitivity, present invention uses the aptamers MUC1 and AS1411 of different proportion (0: 1,
1: 0,1: 1,1: 2,2: 1) optimizing, as a result see that A schemes in Fig. 5.Single aptamer limits MCF-7 cell surface knot
Coincidence point, since detection probe responsiveness reduces, the sensor sheet based on MUC1 AS1411 aptamers reveals relatively
Lower responsiveness (1: 0,0: 1).When using MUC1 and AS1411 aptamers 1: 1, best response has as a result been obtained.This
Outside, 2: 1 or 1: 2 ratio of MUC1 and AS1411, response are below 1: 1 ratio.Based on these discoveries, the present invention is selected
MUC1 and AS1411 is used for further experiment with the preparation of 1: 1 ratio.
The present invention has studied incubation time of the cell on gold electrode, electrode be incubated in cell solution different time (30,
40,50,60 and 70min).B chart is bright in Fig. 5, and peak signal reaches in 60min, therefore selects 60min as optimum cell
Incubation time.The present invention is investigated the analysis that the incubation time (40,50,60,70 and 80min) of nano-probe responds DPV
Performance.C chart is bright in Fig. 5, as nano-probe incubation time increases to 70min signal enhancing from 40min, reaches in 70min
Turning point, therefore, the best nano-probe incubation time of the cell sensor is 70min.
Embodiment five: tumour cell detection process
DNA enzymatic-aptamers-Platinum Nanoparticles-horse-radish peroxidase nano probe of synthesis is then introduced into different cells
On the sensing interface of concentration, in 37 DEG C of reaction 1h.Then electrode is placed in containing 3mM HQ and 1.5mM H2O2PBS solution in,
5min is reacted, differential pulse voltametry scanning is then carried out.Analysis the result shows that, DPV responds (Δ ip) and cell concentration logarithm
20 to 1 × 107Cells/mL. linear related, equation of linear regression is Δ ip (μ A)=- 2.6776+4.1865
1gCcell(cells/mL)(R2=0.9989).Δ ip is peak point current changing value, is expressed as Δ ip=I-I0, wherein I and I0
It is to exist and there is no the DPV peak point currents of the cell sensor of cancer cell respectively.As a result such as the A figure and B figure in Fig. 6.With 3 σ
Method calculate the detection of MCF-7 cell is limited to 6cells/mL.It is thin with HepG2 human liver cancer cell, HCT116 Human colorectal carcinoma
Born of the same parents, three kinds of cell lines of B16 mouse melanoma cells and cell-free solution are as control, using identical method in PBS buffer solution
The selectivity of middle verifying cell sensor, electrochemical signals variation is obvious when discovery only has cell containing MCF-7.In addition, this method
The feasibility of MCF-7 cell in blood is verified, as a result such as the C figure in Fig. 6.
Embodiment six: the recycling of gold electrode
After Electrochemical Detection, the chemical bond between gold electrode and DNA tetrahedron is destroyed using negative voltage pulse, is discharged electric
The MCF-7 cell of pole capture, principle are shown in that A schemes in Fig. 7.It is followed in PBS (10mM, pH 7.4) solution from -0.9 to -1.7V
The scanning of ring voltammetry, scan frequency are 50mV s-1.The MCF-7 cell released from gold electrode is centrifuged in 1000rpm
5min measures cell viability.Cell, which is placed in trypan blue solution (0.04%, w/w), dyes 3min, observation it is dead its under the microscope
Survival condition.Living cells is not colored, and dead cell can dye indigo plant with trypan blue effect since permeability of cell membrane increases
As a result color is shown in that B schemes in Fig. 7.The size of its impedance is detected to determine whether can to gold electrode obtained with Electrode with Electrochemical Impedance Spectroscopy
With recycling, as a result see that C schemes in Fig. 7.
Claims (7)
1. a kind of biosensor for being used to detect tumour cell of repeatable modification, which is characterized in that using gold electrode as inspection
Interface is surveyed, tetrahedron connects double aptamers and is incorporated in gold electrode surfaces by golden sulfide linkage, and nano-probe captures thin by aptamers
Born of the same parents and be connected to cell sensing interface.
2. a kind of biosensor for being used to detect tumour cell of repeatable modification according to claim 1, feature
It is, tetrahedron connects double aptamers (AS1411-A and MUC1-A) and is incorporated in gold electrode surfaces by golden sulfide linkage.
3. a kind of biosensor for being used to detect tumour cell of repeatable modification according to claim 1 or 2, special
Sign is, when ratio 1: 1 of aptamers MUC1 and AS1411 is best.
4. a kind of biosensor for being used to detect tumour cell of repeatable modification according to claim 1, feature
It is, nano-probe set novel metal organic frame PCN-224-Pt, horseradish peroxidase, double aptamers, ferroheme, G-
Tetrad is in one.
5. a kind of repeatable modification for detect tumour cell biosensor the preparation method, which is characterized in that including with
Lower step:
(1) pre-treatment of gold electrode: gold electrode uses Piranha washing lotion to impregnate 15 minutes first, is subsequently placed on polishing cloth and is beaten
Mill, it is sulfuric acid activated until signal stabilization after ultrasonic treatment, then with 0.5M；
(2) tetrahedron connection aptamers fixation: take respectively 10 μM of the single-stranded connection of tetrahedron aptamers AS1411-A, four
In single-stranded AS1411-B, AS1411-C and AS1411-D Yu Sizhi PCR pipe of face body, the trichloroethyl phosphorus of isometric 1mM is respectively added
Acid esters reacts 1h.Above-mentioned solution is mixed in same PCR pipe, and 10min is reacted under the conditions of 95 DEG C, is cooled to 4 DEG C of items at once
10min is reacted under part, the aptamers AS1411 three-dimensional structure of tetrahedron connection can be obtained.The aptamers MUC1 of tetrahedron connection is three-dimensional
The structure same Fa Ke get of MUC1-A, MUC1-B, MUC1-C and MUC1-D.10 μ L, two kinds of adaptation liquid solutions are taken to be added drop-wise to pre-treatment
Gold electrode, react 3h under the conditions of 37 DEG C.Two kinds of aptamers independently fills by Au-S key to be fixed on gold electrode, can it is efficient,
Specifically capture human breast cancer cell line Bcap-37；
The sequence of the aptamers AS1411-A of the single-stranded connection of tetrahedron of the AS1411 three-dimensional structure are as follows: 5 '-ACA TTC
CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ATT TTT TTT
TTG GTG GTG GTG GTT GTG GTG GTG GTG G-3'；
The sequence of the aptamers MUC1-A of the single-stranded connection of tetrahedron of the MUC1 three-dimensional structure are as follows: 5 '-ACA TTC CTA
AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ATT TTT TTT TTG
CAG TTG ATC CTT TGG ATA CCC TGG-3'；
The sequence of the single-stranded B of tetrahedron of the AS1411 three-dimensional structure and MUC1 three-dimensional structure are as follows: 5 '-HS-TAT CAC CAG
GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'；
The sequence of the single-stranded C of tetrahedron of the AS1411 three-dimensional structure and MUC1 three-dimensional structure are as follows: 5 '-HS-TCA ACT GCC
TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3'；
The sequence of the single-stranded D of tetrahedron of the AS1411 three-dimensional structure and MUC1 three-dimensional structure are as follows: 5 '-HS-TTC AGA CTT
AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3'；
(3) fixation of multifunctional nano probe: then by the DNA enzymatic of synthesis (G- tetrad/ferroheme)-bis- aptamers-PCN-
224-Pt particle-horse-radish peroxidase nano probe is introduced on the sensing interface of different cell concentrations, small in 37 DEG C of reactions 1
When.Then electrode is placed in the PBS solution containing 3mM hydroquinone and 1.5mM hydrogen peroxide, reacts 5 minutes, then carries out
Differential pulse voltametry scanning；
The sequence of the AS1411 aptamers are as follows: 5 '-HS- (CH2)6-TTTGGGTAGGGCGGGTTGGG-TTTTTT-
GGTGGTGGTGGTTGTGGTGGTGGTGG-3'；
The sequence of the MUC1 aptamers are as follows: 5 '-HS- (CH2)6-TTTGGGTAGGGCGGGTTGGG-TTTTTT-
GCAGTTGATCCTTTGGATACCCTGG-3'；
(4) the repetition modification of gold electrode: after experiment, applied voltage -1.7~-0.9V destroys Au-S key and regenerates naked
Gold electrode surfaces, for modifying again, repeatedly.
6. the preparation method of the biosensor for detecting tumour cell of repeatable modification according to claim 5,
It is characterized in that, different cell concentrations described in step (3) are 20,1 × 102, 1 × 103, 1 × 104, 1 × 105, 1 × 106With 1 ×
107cells/mL。
7. the preparation method of the biosensor for detecting tumour cell of repeatable modification according to claim 5,
It is characterized in that, buffer described in step (4): 10mM PBS (pH 7.4), scanning speed: 50mV/s, scanning range: -0.9
~-1.7V.
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