CN104655616A - Preparation method and application of electrochemiluminescence aptamer sensor for detecting tumor marker MUC1 - Google Patents
Preparation method and application of electrochemiluminescence aptamer sensor for detecting tumor marker MUC1 Download PDFInfo
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- CN104655616A CN104655616A CN201510033736.3A CN201510033736A CN104655616A CN 104655616 A CN104655616 A CN 104655616A CN 201510033736 A CN201510033736 A CN 201510033736A CN 104655616 A CN104655616 A CN 104655616A
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Abstract
The invention discloses a preparation method and an application of an electrochemiluminescence aptamer sensor for detecting a tumor marker MUC1. The preparation method is characterized by comprising a step of synthesizing magnetic graphene oxide; simultaneously marking an electrochemiluminescence body and an aptamer on the surface of the magnetic graphene oxide material so as to obtain a multifunctional graphene oxide material; and a step of finally dropping and applying 5-10 mu L of multifunctional graphene oxide material to the surface of a magnetic glassy carbon electrode so as to obtain an electrochemiluminescence aptamer sensor for detecting the tumor marker MUC1. The electrochemiluminescence aptamer sensor can be used for determining the concentration of the tumor marker MUC1 in a to-be-detected sample on the basis of a quantitative relation between electrochemiluminescence intensity and the concentration of a tumor marker MUC1 solution, and the sensor has the advantages of being simple and rapid in detection method, sensitive and accurate in detection result, and high in specificity.
Description
Technical field
The present invention relates to a kind of electrochemiluminescence aptamer sensor, especially relating to a kind of preparation method and application thereof of the electrochemiluminescence aptamer sensor for detecting tumor markers MUC1.
Background technology
Global pathogenesis of cancer situation is on the rise in recent years, the patient dying from malignant tumour increases year by year, can realize the early diagnosis and therapy of tumour is reduce the key of mortality ratio, and the clinical diagnosis of current cancer realizes mainly through the content of tumor markers in monitoring human.Tumor markers refers in tumour generation and breeding, the class biochemical substances being produced by tumour cell itself or by host, in-vivo tumour is reacted and produce, comprise tumor correlated albumen class mark, steroids mark, enzyme mark, gene class mark and embryonal antigen etc., can detect in tumor tissues, blood, body fluid or excreta.
MUC1 has another name called Polymorphic epithelial mucin, is a kind of high molecular weight glycoproteins being distributed widely in the various mucomembranous surface of body.MUC1 is mainly expressed in the cell membrane after birth top of multiple organ, tissue epithelial cell under normal circumstances, distribution or polarity distribution in top, but in kinds of tumor cells, the expression appearance amount of MUC1 and the change of matter, and relevant to the infiltration of tumour, Metastasis and prognosis, be the very valuable tumor markers of one.Therefore, simply, quickly and accurately detect tumor markers MUC1, to the early diagnosis of kinds cancer and Index for diagnosis significant.
At present, the method detecting tumor markers MUC1 mainly contains radio immunoassay, fluoroimmunoassay, enzyme-linked immunosorbent assay and Electrical chemiluminescence immunoassay analysis method etc., these methods are all based on antigen-antibody specific binding reaction, highly sensitive, high specificity, but the weak point that also existence one is common, namely antibody be difficult to obtain, high, the poor stability of cost, in addition, said method or there is radiological hazard or operating process is loaded down with trivial details or testing cost is higher.Therefore, develop highly sensitive, high specificity, simply inexpensive in the analytical approach of one, realize the quick detection of tumor markers MUC1, be still current active demand.
Aptamer is filtered out by index concentration Fas lignand system evolution technology (SELEX), by being folded to form artificial single stranded DNA or the RNA sequence of particular space structure and target molecule specific binding, there is high specific, high-affinity, be called " chemical antibody ".As molecular recognition system, compared with antibody, the superiority of aptamer is significant: 1. antibody must use animal or clone to produce molecule, and aptamer is synthesized by in-vitro method and is separated; 2. aptamer easily and can repeat synthesis, and the difference between batch is little, is easy to preserve, and stability, compatibility and specificity are all better than antibody; 3. aptamer has the stability of height to temperature, and antibody is large protein molecule, to responsive to temperature, can produce irreversible sex change.Therefore, aptamer is studied at bio-sensing and Analyze & separate, nucleic acid function, nucleic acid self-assembled material and the research field such as device, disease diagnosis and therapy, shows wide application prospect.
Electrochemiluminescence (ECL) is to immersing the chemiluminescence phenomenon produced containing the electrode of the system of electrochemiluminescence material applies certain voltage or electric current, combine galvanochemistry and chemiluminescent advantage, be a kind of highly sensitive, visual strong, instrument is simple, controllability is strong, the analytical approach of good stability, favorable reproducibility.At present, also do not disclose both at home and abroad any about based on magnetic oxygenated Graphene, electrochemiluminescent and the preparation method of the electrochemiluminescence aptamer sensor for detecting tumor markers MUC1 of aptamer and the correlative study report of application aspect thereof tumor markers MUC1 to specific recognition capability.
Summary of the invention
It is simple that technical matters to be solved by this invention is to provide a kind of assembling process, method of operating is easy, and the sensitive and accuracy of testing result is high, the preparation method of the electrochemiluminescence aptamer sensor for detecting tumor markers MUC1 of high specificity and application thereof.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of preparation method of the electrochemiluminescence aptamer sensor for detecting tumor markers MUC1, comprises the following steps:
(1) synthesis of multifunction graphene oxide composite material
A. the synthesis of magnetic oxygenated Graphene: the graphene oxide (GO) adding 30 ~ 50 mL 1 mg/mL in the three-neck flask of cleaning, ultrasonic 1 h; Then under nitrogen atmosphere protection, add the solution of ferrous chloride of 20 ~ 30 mL 0.05 ~ 0.07 mol/L and the ferric sesquichloride solution of 20 ~ 30 mL 0.10 ~ 0.15 mol/L, in 70 ~ 90 DEG C of stirring and refluxing, slowly drip pH=10 ~ 11 of ammoniacal liquor to solution of 10 ~ 15 mL 20% ~ 30%, reflow treatment 3 ~ 5 h simultaneously; After cooling, wash pH=7 to solution with intermediate water, constant volume, to 50 mL, obtains magnetic oxygenated grapheme material solution, and 4 DEG C store for future use;
B. get the magnetic oxygenated grapheme material solution 200 μ L of gained in step (a) in vial, ultrasonic 1 h, then adds 200 ~ 400 μ L coupling reagents, mix, drip dilute hydrochloric acid solution to pH value of solution=4 ~ 6, oscillation incubation 1 h, intermediate water washs 3 times simultaneously; Add the electrochemiluminescence liquid solution of 30 ~ 50 μ L 0.001 ~ 0.1 mol/L and the aptamers solution of 30 ~ 50 μ L, 10 ~ 20 μm of ol/L again, mix, then drip sodium hydroxide solution to pH value of solution=8 ~ 10, oscillation incubation 4 h, intermediate water washs 3 times; Add bovine serum albumin solution 100 ~ 200 μ L that mass percentage concentration is 1 ~ 2%, 1 h is soaked at 4 DEG C, with the nonspecific activity site on enclosed-electrode surface, intermediate water washs 3 times, be settled to 200 μ L, aptamers and electrochemiluminescent can be obtained with the multifunction graphene oxide composite material solution of tense marker, store for future use at 4 DEG C;
(2) assembling of electrochemiluminescence aptamer sensor
By diameter be the magnetic glassy carbon electrode of 3 ~ 5 mm successively with the alundum (Al2O3) slurry polishing polishing of 1.0 μm, 0.3 μm and 0.05 μm, then ultrasonic 2 min respectively in ethanol and intermediate water, nitrogen dries up; Get the multifunction graphene oxide composite material solution of preparation in 5 ~ 10 μ L steps (1), drip and be coated onto above-mentioned pretreated magnetic glassy carbon electrode surface, namely multifunction graphene oxide composite material evenly, is securely adsorbed onto electrode surface, obtains electrochemiluminescence aptamer sensor.
In described electrochemiluminescence liquid solution, solute is luminol or the different luminol of N-(4-aminobutyl)-N-ethyl, and solvent is 0.1mol/L sodium hydroxide solution.
Described coupling reagent is that 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) obtains with N-hydroxy-succinamide (NHS) is soluble in water, in described coupling reagent, the volumetric molar concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is 10 ~ 100 mmol/L, and described N-hydroxy-succinamide (NHS) volumetric molar concentration is 1 ~ 10 mmol/L.
Described aptamers is amido modified DNA fragmentation, and base sequence is: 5 '-NH
2-GCAGTTGATCCTTTGGATACCCTGG-3 '.
Utilize above-mentioned electrochemiluminescence aptamer sensor to detect the method for tumor markers MUC1, concrete steps are as follows:
The electrochemiluminescence aptamer sensor being used for detecting tumor markers MUC1 of above-mentioned preparation is immersed in the tumor markers MUC1 solution of variable concentrations, incubation 1 h at 37 DEG C, as working electrode after then using intermediate water to wash gently; Use platinum electrode as to electrode, Ag/AgCl electrode or saturated calomel electrode, as contrast electrode, form three-electrode system; Three-electrode system is placed in buffer solution, starts electrochemical reaction, measure the electrochemiluminescence intensity level of system; Obtain the electrochemiluminescence intensity level that the tumor markers MUC1 solution of a series of variable concentrations is corresponding, set up the quantitative relationship between electrochemiluminescence intensity level and tumor markers MUC1 concentration; The concentration of tumor markers MUC1 in testing sample can be detected according to this quantitative relationship.
Described buffer solution is for containing 1 ~ 3 mmol/L H
2o
2the Na of 0.05 mol/L pH=9 ~ 11
2cO
3-NaHCO
3system buffer solution.
The condition of electrochemical reaction is as follows: potential step chronoamperometry; Pulse width: 0.25 second; Recurrent interval: 30 seconds; Initial voltage: 0 V; Pulse voltage: 1 V.
Inventive principle: multifunction graphene oxide composite material is the compound substance being simultaneously modified with ferriferrous oxide nano magnetic bead, aptamers and electrochemiluminescent on graphene oxide, there is several functions: (1) graphene oxide has that specific surface area is large, surface functional group is many and the advantage such as good biocompatibility, therefore can a large amount of ferriferrous oxide nano magnetic bead, aptamers and the electrochemiluminescent of load; (2) ferriferrous oxide nano magnetic bead not only has superpower magnetic, multifunction graphene oxide composite material is made evenly, to be securely adsorbed onto electrode surface, realize a step assembling of electrochemiluminescence aptamer sensor, simplify the preparation method of sensor, also there is excellent electric conductivity simultaneously, strengthen the electric conductivity of multifunction graphene oxide composite material, promote the electron transfer process of electrode surface; (3) aptamers has specific recognition capability to tumor markers MUC1, can identify specifically and catch the tumor markers MUC1 in solution; (4) electrochemiluminescent can produce strong and stable electrochemical luminescence signals in electrochemical reaction.
The present invention successfully can build the electrochemiluminescence aptamer sensor detecting tumor markers MUC1 after multifunction graphene oxide composite material being dripped and being coated onto magnetic glassy carbon electrode surface, this sensor, under electrochemical signals excites, can produce strong and stable electrochemiluminescence; If containing tumor markers MUC1 in testing sample, MUC1 will be caught by the aptamers in electrochemiluminescence aptamer sensor, due to the protein that MUC1 is a kind of high molecular, therefore being attached to electrode surface can the very big electron transmission on impeded electrode surface and the transmitting procedure of light, causes electrochemiluminescence intensity to reduce; The concentration of tumor markers MUC1 is larger, and electrochemiluminescence intensity level is lower, linear between the log concentration of electrochemiluminescence intensity level and tumor markers MUC1, can realize the quantitative detection of tumor markers MUC1 in testing sample accordingly.The preparation flow of the electrochemiluminescence aptamer sensor of tumor markers MUC1 and Cleaning Principle figure is detected as shown in Figure 1 based on multifunction graphene oxide composite material.
Compared with prior art, the invention has the advantages that
(1) highly sensitive.The detectability that electrochemiluminescence aptamer sensor prepared by the present invention detects tumor markers MUC1 reaches 0.001 U/mL, and the detectability of existing detection method is generally 0.01 ~ 200 U/mL.Reason is: Electrochemiluminescence technology itself has high sensitivity, and this sensor has unique structural design and improves sensitivity further.
(2) high specificity.Other common tumor markerses are all noiseless to this detection system.Reason is: the present invention is the electrochemiluminescence aptamer sensor built based on having the high specific binding ability between the aptamers of specific recognition capability and tumor markers MUC1 to tumor markers MUC1, other tumor markers except MUC1 is not the target substance of aptamers, therefore other tumor markerses in testing sample all can not be caught by the aptamers in electrochemiluminescence aptamer sensor, therefore noiseless to detection system.
(3) testing result is accurate.Detect the recovery of standard addition > 96% of actual sample.
(4) sensor assembling process is simple, and testing process is quick.Multifunction graphene oxide composite material dripped and be coated onto magnetic glassy carbon electrode surface, successfully can be built the electrochemiluminescence aptamer sensor of this method by this single stepping, assembling process is extremely simple; After the electrochemiluminescence aptamer sensor assembled and testing sample solution have been hatched, electrochemiluminescence can be carried out and detect the corresponding electrochemical luminescence signals value of acquisition, get final product the quantitative detection of realize target thing according to linear equation.
In sum, the present invention successfully constructs a kind of electrochemiluminescence aptamer sensor detecting tumor markers MUC1 based on multifunction graphene oxide composite material by a step assembling process, this sensor has the high specific of aptamers analysis and the high sensitivity of Electrochemiluminescence technology concurrently, have highly sensitive, selectivity good, simple to operate, analyze the advantages such as quick, with low cost, can realize, to the detection of super low concentration tumor markers MUC1, having a good application prospect.
Accompanying drawing explanation
Fig. 1 is the preparation flow and the Cleaning Principle figure that detect the electrochemiluminescence aptamer sensor of tumor markers MUC1 based on multifunction graphene oxide composite material;
Fig. 2 is the preparation flow figure of multifunction graphene oxide composite material;
Fig. 3 be detect variable concentrations MUC1 electrochemical luminescence signals intensity level (
y)-concentration (
x) linear graph of logarithm;
Fig. 4 is that the present invention is for detecting the selectivity figure of the electrochemiluminescence aptamer sensor of tumor markers MUC1.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment one
For detecting a preparation method for the electrochemiluminescence aptamer sensor of tumor markers MUC1, comprise the following steps:
(1) synthesis of multifunction graphene oxide composite material, its preparation flow as shown in Figure 1
A. the synthesis of magnetic oxygenated Graphene: the graphene oxide (GO) adding 40 mL 1 mg/mL in the three-neck flask of cleaning, ultrasonic 1 h(ultrasound wave process 1 h, general power about the 200W used); Then under nitrogen atmosphere protection, add the solution of ferrous chloride of 25 mL 0.06 mol/L and the ferric sesquichloride solution of 25 mL 0.12 mol/L, in 80 DEG C of stirring and refluxing, slowly drip pH=10.5 of ammoniacal liquor to solution of 12 mL 25%, reflow treatment 4 h simultaneously; After cooling, wash pH=7 to solution with intermediate water, constant volume, to 50 mL, obtains magnetic oxygenated grapheme material solution, and 4 DEG C store for future use;
B. get the magnetic oxygenated grapheme material solution 200 μ L of gained in step (a) in vial, ultrasonic 1 h, then adds 300 μ L coupling reagents, mix, drip dilute hydrochloric acid solution to pH value of solution=5, oscillation incubation 1 h, intermediate water washs 3 times simultaneously; (solute is luminol to add the electrochemiluminescence liquid solution of 40 μ L 0.01 mol/L again, solvent is 0.1mol/L sodium hydroxide solution) and the aptamers solution (DNA fragmentation is water-soluble formulated) of 40 μ L, 15 μm of ol/L, mix, then sodium hydroxide solution is dripped to pH value of solution=9, oscillation incubation 4 h, after intermediate water washs 3 times; Add the bovine serum albumin solution 150 μ L that mass percentage concentration is 1.5%, 1 h is soaked at 4 DEG C, with the nonspecific activity site on enclosed-electrode surface, intermediate water washs 3 times, be settled to 200 μ L, aptamers and electrochemiluminescent can be obtained with the multifunction graphene oxide composite material solution of tense marker, store for future use at 4 DEG C; Wherein coupling reagent is that 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) obtains with N-hydroxy-succinamide (NHS) is soluble in water, in coupling reagent, the volumetric molar concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is 50 mmol/L, and N-hydroxy-succinamide (NHS) volumetric molar concentration is 5mmol/L; Aptamers is amido modified DNA fragmentation, and base sequence is: 5 '-NH
2-GCAGTTGATCCTTTGGATACCCTGG-3 ';
(2) assembling of electrochemiluminescence aptamer sensor
By diameter be the magnetic glassy carbon electrode of 3 ~ 5 mm successively with the alundum (Al2O3) slurry polishing polishing of 1.0 μm, 0.3 μm and 0.05 μm, then ultrasonic 2 min respectively in ethanol and intermediate water, nitrogen dries up; Get the multifunction graphene oxide composite material solution of preparation in 8 μ L steps (1), drip and be coated onto above-mentioned pretreated magnetic glassy carbon electrode surface, namely multifunction graphene oxide composite material evenly, is securely adsorbed onto electrode surface, obtains electrochemiluminescence aptamer sensor.
Specific embodiment two
With above-described embodiment one, its key distinction is:
In a step of step (1): the graphene oxide adding 30 mL 1 mg/mL in the three-neck flask of cleaning, ultrasonic 1 h; Then under nitrogen atmosphere protection, add the solution of ferrous chloride of 20 mL 0.07 mol/L and the ferric sesquichloride solution of 20 mL 0.15 mol/L, in 70 DEG C of stirring and refluxing, slowly drip pH=10 of ammoniacal liquor to solution of 10 mL 30%, reflow treatment 5 h simultaneously; After cooling, wash pH=7 to solution with intermediate water, constant volume, to 50 mL, obtains magnetic oxygenated grapheme material solution;
In the b step of step (1), get magnetic oxygenated grapheme material solution ultrasonic in vial, then add 200 μ L coupling reagents, drip dilute hydrochloric acid solution to pH value of solution=4, oscillation incubation 1 h, intermediate water washs 3 times simultaneously; (solute is the different luminol of N-(4-aminobutyl)-N-ethyl to add the electrochemiluminescence liquid solution of 30 μ L 0.1 mol/L again, solvent is 0.1mol/L sodium hydroxide solution) and the aptamers solution of 30 μ L, 20 μm of ol/L, then sodium hydroxide solution is dripped to pH value of solution=8, oscillation incubation 4 h, after intermediate water washs 3 times; Add the bovine serum albumin solution 200 μ L that mass percentage concentration is 1%, soak 1 h at 4 DEG C, intermediate water washs 3 times, is settled to 200 μ L, can obtain aptamers and the electrochemiluminescent multifunction graphene oxide composite material solution with tense marker; Wherein in coupling reagent, the volumetric molar concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is 10 mmol/L, and described N-hydroxy-succinamide (NHS) volumetric molar concentration is 10 mmol/L;
Get 5 μ L multifunction graphene oxide composite material solution in step (2) to drip and be coated onto magnetic glassy carbon electrode surface.
Specific embodiment three
With above-described embodiment one, its key distinction is:
In a step of step (1): the graphene oxide adding 50 mL 1 mg/mL in the three-neck flask of cleaning, ultrasonic 1 h; Then under nitrogen atmosphere protection, add the solution of ferrous chloride of 30 mL 0.05 mol/L and the ferric sesquichloride solution of 30 mL 0.10 mol/L, in 90 DEG C of stirring and refluxing, slowly drip pH=11 of ammoniacal liquor to solution of 15 mL 20%, reflow treatment 3 h simultaneously; After cooling, wash pH=7 to solution with intermediate water, constant volume, to 50 mL, obtains magnetic oxygenated grapheme material solution;
In the b step of step (1), get magnetic oxygenated grapheme material solution ultrasonic in vial, then add 400 μ L coupling reagents, mix, drip dilute hydrochloric acid solution to pH value of solution=6, oscillation incubation 1 h, intermediate water washs 3 times simultaneously; Add the Particles in Electrochemiluminescofce ofce Luminol liquid solution of 50 μ L 0.001 mol/L and the aptamers solution of 50 μ L, 10 μm of ol/L again, then drip sodium hydroxide solution to pH value of solution=10, oscillation incubation 4 h, after intermediate water washs 3 times; Add the bovine serum albumin solution 100 μ L that mass percentage concentration is 2%, soak 1 h at 4 DEG C, intermediate water washs 3 times, is settled to 200 μ L, can obtain aptamers and the electrochemiluminescent multifunction graphene oxide composite material solution with tense marker; Wherein in coupling reagent, the volumetric molar concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is 100 mmol/L, and described N-hydroxy-succinamide (NHS) volumetric molar concentration is 1 mmol/L;
Get 10 μ L multifunction graphene oxide composite material solution in step (2) to drip and be coated onto magnetic glassy carbon electrode surface.
Specific embodiment four
A method of the detection tumor markers MUC1 of the electrochemiluminescence aptamer sensor for detecting tumor markers MUC1 utilizing above-mentioned specific embodiment one to prepare, concrete steps are as follows:
The electrochemiluminescence aptamer sensor being used for detecting tumor markers MUC1 prepared by specific embodiment one is immersed in the tumor markers MUC1 solution of variable concentrations, incubation 1 h at 37 DEG C, as working electrode after then using intermediate water to wash gently; Use platinum electrode as to electrode, Ag/AgCl electrode or saturated calomel electrode, as contrast electrode, form three-electrode system; Three-electrode system is placed in buffer solution, starts electrochemical reaction, measure the electrochemiluminescence intensity level of system; Obtain the electrochemiluminescence intensity level that the tumor markers MUC1 solution of a series of variable concentrations is corresponding, set up the quantitative relationship between electrochemiluminescence intensity level and tumor markers MUC1 concentration; When the concentration of MUC1 is 0.001 ~ 100 U/mL, present good linear relationship as shown in Figure 3 between electrochemical luminescence signals intensity level and the log concentration of MUC1, linear equation is
y=– 1997.38 × log
x+ 4979.25, linearly dependent coefficient
r=0.9948.Can the concentration of tumor markers MUC1 in accurate quantitative analysis unknown sample according to linear equation.
Above-mentioned buffer solution is for containing 1 ~ 3 mmol/L H
2o
2the Na of 0.05 mol/L pH=9 ~ 11
2cO
3-NaHCO
3system buffer solution.
The condition of above-mentioned electrochemical reaction is as follows: potential step chronoamperometry; Pulse width: 0.25 second; Recurrent interval: 30 seconds; Initial voltage: 0 V; Pulse voltage: 1 V.
Specific embodiment five
Electrochemiluminescence aptamer sensor specific test
Experiment have chosen other common tumor markers neurotensins (NT), human immunoglobulin (hIgG), alpha-fetoprotein (AFP) and carcinomebryonic antigen (CEA) verify the specific embodiment of the invention one, the electrochemiluminescence aptamer sensor of two and three preparations has specific recognition capability to tumor markers MUC1, result shows as shown in Figure 4: when other common tumor markers exist, the signal of this electrochemiluminescence aptamer sensor is roughly suitable with blank value, this shows that the detection of common tumor markers to MUC1 is disturbed without conspicuousness, selectivity is good.Wherein the concentration of NT, hIgG, AFP and CEA is 1000U/mL, MUC1 concentration is 10 U/mL.
Specific embodiment six
Get blank serum samples, adopt standard addition method to prepare the MUC1 solution of variable concentrations respectively, construct electrochemiluminescence aptamer sensor according to the specific experiment step in invention specific embodiment one, two and three and mark-on sample is detected by specific embodiment four method.Testing result is as shown in table 1,
In table 1 human serum tumor markers MUC1 detection (
,
n=5)
| Blood serum sample | Add scalar/(U/mL) | Detected level/(U/mL) | RSD/% | The recovery/% |
| 1 | 100 | 97.23 ± 2.2 | 2.3 | 97.2% |
| 2 | 50 | 51.2 ± 0.82 | 1.6 | 102.4% |
| 3 | 10 | 9.83 ± 0.25 | 2.5 | 98.3% |
| 4 | 5 | 5.08 ± 0.14 | 2.7 | 101.6% |
| 5 | 1 | 0.961 ±0.03 | 3.1 | 96.1% |
As shown in Table 1, relative standard deviation (RSD) is less than 3.1%, and the recovery is between 96.1 ~ 102.4%, and show that the detection precision of electrochemiluminescence aptamer sensor to serum tumor mark MUC1 that the present invention builds is high, testing result accurately and reliably.
These results suggest that, the electrochemiluminescence aptamer sensor of detection tumor markers MUC1 that the present invention builds, highly sensitive, detectability is low, selectivity good, simple to operate, testing result accurately and reliably.Only multifunction graphene oxide composite material need be dripped in experimentation and be coated onto the step assembling that magnetic glassy carbon electrode surface can realize electrochemiluminescence aptamer sensor, and then realize high sensitivity, strong specificity to serum tumor mark MUC1, simply, fast detect.
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned citing.Those skilled in the art are in essential scope of the present invention, and the change made, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.
Claims (7)
1., for detecting a preparation method for the electrochemiluminescence aptamer sensor of tumor markers MUC1, it is characterized in that comprising the following steps:
(1) synthesis of multi-functional graphene oxide composite material
A. the synthesis of magnetic oxygenated Graphene: the graphene oxide adding 30 ~ 50 mL 1 mg/mL in the three-neck flask of cleaning, ultrasonic 1 h; Then under nitrogen atmosphere protection, add the solution of ferrous chloride of 20 ~ 30 mL 0.05 ~ 0.07 mol/L and the ferric sesquichloride solution of 20 ~ 30 mL 0.10 ~ 0.15 mol/L, in 70 ~ 90 DEG C of stirring and refluxing, slowly drip pH=10 ~ 11 of ammoniacal liquor to solution of 10 ~ 15 mL 20% ~ 30%, reflow treatment 3 ~ 5 h simultaneously; After cooling, wash pH=7 to solution with intermediate water, constant volume, to 50 mL, obtains magnetic oxygenated grapheme material solution, and 4 DEG C store for future use;
B. get the magnetic oxygenated grapheme material solution 200 μ L of gained in step (a) in vial, ultrasonic 1 h, then adds 200 ~ 400 μ L coupling reagents, mix, drip dilute hydrochloric acid solution to pH value of solution=4 ~ 6, oscillation incubation 1 h, intermediate water washs 3 times simultaneously; Add the electrochemiluminescence liquid solution of 30 ~ 50 μ L 0.001 ~ 0.1 mol/L and the aptamers solution of 30 ~ 50 μ L, 10 ~ 20 μm of ol/L again, mix, then drip sodium hydroxide solution to pH value of solution=8 ~ 10, oscillation incubation 4 h, after intermediate water washs 3 times; Add bovine serum albumin solution 100 ~ 200 μ L that mass percentage concentration is 1 ~ 2%, soak 1 h at 4 DEG C, intermediate water washs 3 times, is settled to 200 μ L, aptamers and electrochemiluminescent can be obtained with the multifunction graphene oxide composite material solution of tense marker, store for future use at 4 DEG C;
(2) assembling of electrochemiluminescence aptamer sensor
By diameter be the magnetic glassy carbon electrode of 3 ~ 5 mm successively with the alundum (Al2O3) slurry polishing polishing of 1.0 μm, 0.3 μm and 0.05 μm, then ultrasonic 2 min respectively in ethanol and intermediate water, nitrogen dries up; Get the multifunction graphene oxide composite material solution of preparation in 5 ~ 10 μ L steps (1), drip and be coated onto above-mentioned pretreated magnetic glassy carbon electrode surface, namely multifunction graphene oxide composite material evenly, is securely adsorbed onto electrode surface, obtains electrochemiluminescence aptamer sensor.
2. the preparation method of the electrochemiluminescence aptamer sensor for detecting tumor markers MUC1 according to claim 1, it is characterized in that: in described electrochemiluminescence liquid solution, solute is luminol or the different luminol of N-(4-aminobutyl)-N-ethyl, and solvent is 0.1mol/L sodium hydroxide solution.
3. the preparation method of the electrochemiluminescence aptamer sensor for detecting tumor markers MUC1 according to claim 1, it is characterized in that: described coupling reagent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide is soluble in water obtains, in described coupling reagent, the volumetric molar concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is 10 ~ 100 mmol/L, and described N-hydroxy-succinamide volumetric molar concentration is 1 ~ 10 mmol/L.
4. the preparation method of the electrochemiluminescence aptamer sensor for detecting tumor markers MUC1 according to claim 1, is characterized in that: described aptamers is amido modified DNA fragmentation, and base sequence is: 5 '-NH
2-GCAGTTGATCCTTTGGATACCCTGG-3 '.
5. utilize the electrochemiluminescence aptamer sensor according to any one of claim 1-4 to detect a method of tumor markers MUC1, it is characterized in that concrete steps are as follows:
The electrochemiluminescence aptamer sensor being used for detecting tumor markers MUC1 prepared any one of claim 1-4 is immersed in the tumor markers MUC1 solution of variable concentrations, incubation 1 h at 37 DEG C, as working electrode after then using intermediate water to wash gently; Use platinum electrode as to electrode, Ag/AgCl electrode or saturated calomel electrode, as contrast electrode, form three-electrode system; Three-electrode system is placed in buffer solution, starts electrochemical reaction, measure the electrochemiluminescence intensity level of system; Obtain the electrochemiluminescence intensity level that the tumor markers MUC1 solution of a series of variable concentrations is corresponding, set up the quantitative relationship between electrochemiluminescence intensity level and tumor markers MUC1 concentration; The concentration of tumor markers MUC1 in testing sample can be detected according to this quantitative relationship.
6. electrochemiluminescence aptamer sensor according to claim 5 detects the method for tumor markers MUC1, it is characterized in that: described buffer solution is for containing 1 ~ 3 mmol/L H
2o
2the Na of 0.05 mol/L pH=9 ~ 11
2cO
3-NaHCO
3system buffer solution.
7. electrochemiluminescence aptamer sensor according to claim 5 detects the method for tumor markers MUC1, it is characterized in that the condition of electrochemical reaction is as follows: potential step chronoamperometry; Pulse width: 0.25 second; Recurrent interval: 30 seconds; Initial voltage: 0 V; Pulse voltage: 1 V.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105067597A (en) * | 2015-07-23 | 2015-11-18 | 江苏大学 | Electrochemical luminescence method for detecting graphene oxide |
| CN105241869A (en) * | 2015-09-29 | 2016-01-13 | 江南大学 | Bisphenol A electrochemiluminescent aptamer sensor based on upper conversion nano material |
| CN105806905A (en) * | 2016-03-10 | 2016-07-27 | 中南大学 | Methylene blue probe based biosensor capable of simultaneously detecting two tumor markers and building method and application of biosensor |
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| CN106248658A (en) * | 2016-09-08 | 2016-12-21 | 济南大学 | The preparation of paper substrate electrochemiluminescence converter and the application in carcinoembryonic antigen detects |
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| CN105067597A (en) * | 2015-07-23 | 2015-11-18 | 江苏大学 | Electrochemical luminescence method for detecting graphene oxide |
| CN105241869A (en) * | 2015-09-29 | 2016-01-13 | 江南大学 | Bisphenol A electrochemiluminescent aptamer sensor based on upper conversion nano material |
| CN106610375A (en) * | 2015-10-23 | 2017-05-03 | 复旦大学 | Oxidized mesoporous carbon nanosphere aptasensor, and preparation method and application thereof |
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| CN108828032B (en) * | 2018-09-18 | 2024-04-19 | 天津博硕科技有限公司 | Online electrochemical detection device, method and electrochemical immunoassay instrument |
| CN109725041A (en) * | 2018-12-24 | 2019-05-07 | 江苏大学 | A kind of preparation method and application of bisphenol-A electrochemical luminescence aptamer sensor |
| CN109856210A (en) * | 2019-01-30 | 2019-06-07 | 西北大学 | A kind of electrochemical luminescence biosensor and preparation method thereof detecting early stage of lung cancer marker NAP-2 albumen |
| CN110514711A (en) * | 2019-08-17 | 2019-11-29 | 山东理工大学 | The preparation method of renewable aptamer sensor based on automatically cleaning electrode interface |
| CN110514711B (en) * | 2019-08-17 | 2021-12-21 | 山东理工大学 | Preparation method of renewable aptamer sensor based on self-cleaning electrode interface |
| CN113063761A (en) * | 2021-03-17 | 2021-07-02 | 北京化工大学 | Fluorescent aptamer sensor for detecting muc1 mucin and application method thereof |
| CN113063761B (en) * | 2021-03-17 | 2023-02-10 | 北京化工大学 | Fluorescent aptamer sensor for detecting muc1 mucin and application method thereof |
| CN113624824A (en) * | 2021-08-05 | 2021-11-09 | 青岛农业大学 | Preparation method and application of sensor based on nano composite material |
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