CN103940872B - Detect preparation method and the application of two kinds of acute leukemia mark electrochemical sensors simultaneously - Google Patents
Detect preparation method and the application of two kinds of acute leukemia mark electrochemical sensors simultaneously Download PDFInfo
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Abstract
The invention discloses a kind of preparation method simultaneously detecting two kinds of acute leukemia mark electrochemical sensors, step is as follows: lysozyme reporter probe and gamma interferon reporter probe left standstill in containing the solution of TCEP respectively, with opened disulfide bond and respectively and gamma interferon fit formation duplex structure fit with lysozyme; Pre-service is carried out to gold electrode, the gold electrode handled well is immersed in the mixed liquor of pretreated lysozyme reporter probe-fit double-strand and gamma interferon reporter probe-fit double-strand, room temperature hold over night, afterwards with redistilled water and cleaning fluid cleaning; Then electrode is immersed in the solution containing MCH, with enclosed-electrode, afterwards with redistilled water and cleaning fluid cleaning; Using the electrode after above-mentioned steps process as working electrode, and contrast electrode, to Electrode connection on chem workstation, to obtain electrochemical sensor.This electrochemical sensor detects while can be used for two kinds of acute leukemia mark lysozymes and gamma interferon.
Description
Technical field
The present invention relates to a kind of preparation method and the application that detect two kinds of acute leukemia mark electrochemical sensors simultaneously, belong to clinical analysis and bioelectrochemical sensing technical field.
Background technology
In society, cancer is one of disease of serious threat human body health.The topmost reason that the mortality ratio of cancer patient is very high to carry out early diagnosis, if can early diagnosis treat in time, the cancer of 1/3 can be cured.And the early diagnosis of the mensuration of blood serum tumor markers to cancer plays an important role.Therefore, foundation analytical approach that is accurate, detection serum tumor mark fast and corresponding equipment cause the great attention of people.
Acute leukemia is modal a kind of malignant tumour in children and youth, very harmful to children's torso health, and its early diagnosis is of great significance effective treatment tool.Change in traditional diagnosis method many dependence tumor tissues or cellular morphology is carried out, but judges that difficulty is large, accuracy rate is low, and once determine that cancer occurs on tissue and cellular morphology, often enters middle and advanced stage, be difficult to effectively treat.Because the process of cancer cell change is mainly induced by the change of the molecular level of complexity, therefore study the difference on molecular level, sensitive and specific recognition and detection are carried out to the significant material of this level, is expected to the efficient early diagnosis realizing cancer.
Summary of the invention
The object of the present invention is to provide a kind of preparation method and the application that detect two kinds of acute leukemia mark electrochemical sensors simultaneously.
The technology used in the present invention solution is:
Detect a preparation method for two kinds of acute leukemia mark electrochemical sensors simultaneously, comprise the following steps:
(1) lysozyme reporter probe and gamma interferon reporter probe are left standstill, with opened disulfide bond respectively in containing the solution of three (2-chloroethyl) phosphate; Lysozyme is added again fit fit with gamma interferon in solution, lysozyme reporter probe and lysozyme fit formation lysozyme reporter probe-fit duplex structure, gamma interferon reporter probe and gamma interferon fit formation gamma interferon reporter probe-fit duplex structure;
(2) pre-service is carried out to gold electrode, the gold electrode handled well is immersed in step (1) pretreated lysozyme reporter probe-fit double-strand and gamma interferon reporter probe-fit double-strand mixed liquor, room temperature hold over night, afterwards with redistilled water and cleaning fluid cleaning;
(3) electrode after step (2) being processed is immersed in the solution containing 6-sulfydryl-1-hexanol, with enclosed-electrode, afterwards with redistilled water and cleaning fluid cleaning;
(4) using step (3) process after electrode as working electrode, and contrast electrode, to Electrode connection on chem workstation, to obtain the electrochemical sensor that can detect two kinds of acute leukemia marks simultaneously.
Preferably, in step (1): described lysozyme reporter probe is 5 '-sulfydryl-C6-GTGCAGAGCTAAGTAACTCTGCAC-methylene blue-3 ', described lysozyme is fit is 5 '-ATCAGGGCTAAAGAGTGCAGAGTTACTTAG-3 ', described gamma interferon reporter probe is 5 '-sulfydryl-C6-TTCAACCAACTTTTTTTTTTCAACACCCAT-ferrocene-3 ', described gamma interferon is fit is 5 '-GGGGTTGGTTGTGTTGGGTGTTGTGT-3 '.
Preferably, in step (1): the concentration of described Lysozyme in Solution reporter probe-fit double-strand is 1 μm of ol/L, the concentration of gamma interferon reporter probe-fit double-strand is 1 μm of ol/L; Containing 0.5 μ L10mmol/L tri-(2-chloroethyl) phosphate in described solution, also containing 20mmol/LTris-HCl, 140mmol/LNaCl, 20mmol/LMgCl
2and 20mmol/LKCl, solution ph is 7.4.
Preferably, in step (1): described standing process is at room temperature carried out, time of repose is 2 hours.
Preferably, described in step (2), the preprocessing process of gold electrode is as follows: the gold electrode Al of 0.3 μm
2o
3powder is polished into minute surface, after redistilled water cleaning, and ultrasonic 50s in redistilled water and ethanolic solution in succession; By the gold electrode cleaned up, in the sulfuric acid solution of 0.5mol/L, cyclic voltammetry scan is to stablizing, and scanning voltage is 0.2 ~ 1.65V, and sweep velocity is 0.1V/s, and then rinse well with redistilled water, room temperature is dried.
Preferably, in step (3): in described solution, the concentration of MCH is 2mmol/L, also containing 20mmol/LTris-HCl, 140mmol/LNaCl, 20mmol/LMgCl in solution
2and 20mmol/LKCl, solution ph is 7.4.
Preferably, step (2) is with step (3): described cleaning fluid is the Tris-HCl buffer solution of concentration 10mmol/L, and solution ph is 7.4.
The electrochemical sensor of above-mentioned preparation is for detecting while two kinds of acute leukemia mark lysozymes and gamma interferon.
Above-mentioned detection method specifically comprises the following steps:
(1) lysozyme and gamma interferon detect the preparation of solution: lysozyme and gamma interferon are mixed with concentration in the solution and are respectively 0.5 ~ 500nmol/L and 0.5 ~ 500nmol/L, for subsequent use at being stored in 4 DEG C; 20mmol/LTris-HCl, 140mmol/LNaCl, 20mmol/LMgCl is included in described solution
2and 20mmol/LKCl, solution ph is 7.4;
(2) prepared working sensor electrode is immersed in lysozyme that 50 μ L steps (1) prepare and gamma interferon detects in solution, leaves standstill 1h at 37 DEG C, afterwards with redistilled water and cleaning fluid cleaning, obtain the working electrode assembled;
(3) by contrast electrode, be connected on electrochemical workstation to the working electrode of electrode and assembling, the solution of 40 ~ 60 μ L is added in a cell, containing 20mmol/LTris-HCl, 140mmol/LNaCl, 20mmol/LMgCl in solution
2and 20mmol/LKCl, solution ph is 7.4, then detects the current-responsive of the working electrode of assembling with differential pulse voltammetry, according to the relation between gained current-responsive and the concentration of standard solution of two kinds of acute leukemia marks, and drawing curve;
(4) testing sample solution is replaced the standard solution of two kinds of marks, detect according to the method for drafting income of the working curve of described two kinds of acute leukemia marks.
Preferably, in step (2), described cleaning fluid is the Tris-HCl buffer solution of concentration 10mmol/L, and pH value of solution is 7.4.
Principle of work of the present invention and beneficial effect as follows:
At the ill initial stage, the mark that lysozyme, IFN-γ etc. are relevant to acute leukemia can change, and carrying out early diagnosis and somatotype, having very large meaning to the early diagnosis of acute leukemia to detecting to assist while them.The present invention is based on aptamer recognition technology, set up electrochemica biological sensor realize lysozyme (its concentration contribute to distinguish the hypotype such as acute promyelocytic leukemia, acute myelomonocytic leukemia) and IFN-γ (its concentration can reflect cellular immune level and the body's immunity of acute leukemic patient) two kinds of acute leukemia related substanceses while, highly sensitive detection, for acute leukemia early diagnosis.
First the present invention fixes reporter probe-fit double-strand that two kinds are modified with the mark of electroactive material (ferrocene, methylene blue) respectively on gold electrode, under lysozyme and gamma interferon exist both respectively with the fit combination in double-strand, the conformation of two reporter probes changes respectively thus causes the distance between electroactive material and electrode that corresponding reporter probe chain is modified to change, thus current-responsive changes.The present invention is in conjunction with fit recognition technology, and prepared sensor has high selectivity, can realize the high-sensitivity detection of lysozyme and IFN-γ simultaneously; Only need during detection sample simple process, save detection time, reduce testing cost, be a kind of quick, cheap and sensitive detection method, will the development of acute leukemia getting up early diagnosis and somatotype be promoted.
Accompanying drawing explanation
Fig. 1 is schematic diagram of the present invention.
Embodiment
By reference to the accompanying drawings, a kind of preparation method simultaneously detecting two kinds of acute leukemia mark electrochemical sensors, comprises the following steps:
(1) by 1 μm of ol/L lysozyme reporter probe (5 '-sulfydryl-C6-GTGCAGAGCTAAGTAACTCTGCAC-methylene blue-3 ') and 1 μm of ol/L gamma interferon reporter probe (5 '-sulfydryl-C6-TTCAACCAACTTTTTTTTTTCAACACCCAT-ferrocene-3 ') respectively at the 50 μ L solution (20mmol/LTris-HCl containing 0.5 μ L10mmol/L tri-(2-chloroethyl) phosphate (TCEP), pH7.4,140mmol/LNaCl, 20mmol/LMgCl2, left at room temperature 2h 20mmol/LKCl), with opened disulfide bond.1 μm of ol/L lysozyme fit (5 '-ATCAGGGCTAAAGAGTGCAGAGTTACTTAG-3 ') is added again and 1 μm of ol/L interferon fit (5 '-GGGGTTGGTTGTGTTGGGTGTTGTGT-3 ') in solution, lysozyme reporter probe and lysozyme fit formation lysozyme reporter probe-fit duplex structure, gamma interferon reporter probe and gamma interferon fit formation gamma interferon reporter probe-fit duplex structure, obtain the fit and fit mixed liquor of gamma interferon of pretreated lysozyme.
(2) the gold electrode Al of 0.3 μm
2o
3powder is polished into minute surface, after redistilled water cleaning, and ultrasonic 50s in redistilled water and ethanolic solution in succession; By the gold electrode cleaned up, in the sulfuric acid solution of 0.5mol/L, cyclic voltammetric (0.2 ~ 1.65V, 0.1V/s) is to stablizing, and then rinse well with redistilled water, room temperature is dried.The gold electrode handled well is immersed in and the fit mixed liquor of gamma interferon fit through step (1) pretreated lysozyme, room temperature hold over night, use redistilled water and cleaning fluid (10mmol/LTris-HCl, pH7.4) cleaning afterwards.
(3) electrode after step (2) process is immersed in solution (20mmol/LTris-HCl, pH7.4,140mmol/LNaCl, the 20mmol/LMgCl containing 2mmol/L6-sulfydryl-1-hexanol (MCH)
2, 20mmol/LKCl) and middle 1h, with enclosed-electrode, uses redistilled water and cleaning fluid (10mmol/LTris-HCl, pH7.4) cleaning afterwards.
(4) using step (3) process after electrode as working electrode, and contrast electrode, to Electrode connection on chem workstation, to obtain the electrochemical sensor that can detect two kinds of acute leukemia marks simultaneously.
Above-mentioned electrochemical sensor detects while can be used for two kinds of acute leukemia mark lysozymes and gamma interferon, specifically comprises the following steps:
(1) lysozyme and gamma interferon detect the preparation of solution: lysozyme and gamma interferon are at solution (20mmol/LTris-HCl, pH7.4,140mmol/LNaCl, 20mmol/LMgCl
2, 20mmol/LKCl) in be mixed with the mixed liquor solution that concentration is respectively 0.5 ~ 500nmol/L and 0.5 ~ 500nmol/L, for subsequent use at being stored in 4 DEG C.
(2) prepared working sensor electrode is immersed in the mixed liquor of the concentration known that 50 μ L steps (1) are prepared, 1h is left standstill at 37 DEG C, use redistilled water and cleaning fluid (10mmol/LTris-HCl afterwards, pH7.4) clean, obtain the working electrode assembled.
(3) by contrast electrode, be connected on electrochemical workstation to the working electrode of electrode and assembling, solution (20mmol/LTris-HCl, pH7.4,140mmol/LNaCl, the 20mmol/LMgCl of 40 ~ 60 μ L is added in a cell
2, 20mmol/LKCl), the current-responsive of the working electrode of assembling is then detected with differential pulse voltammetry, according to the relation between gained current-responsive and the concentration of standard solution of two kinds of acute leukemia marks, drawing curve.
(4) testing sample solution is replaced the standard solution of two kinds of marks, detect according to the method for drafting income of the working curve of described two kinds of acute leukemia marks.
Testing result is as follows:
1, the detection of gamma interferon: the signal of ferrocene is at about 400 ± 4mV, and sensing range is approximately 0.5 ~ 500nmol/L, and detectability is about 0.3nmol/L.
2, the detection of lysozyme: the signal of methylene blue is at about-245 ± 3mV, and sensing range is approximately 0.5 ~ 500nmol/L detectability and is about 0.2nmol/L.
Can find out by detecting data above, sensor of the present invention can realize high-sensitivity detection simultaneously to lysozyme and interferon.
Claims (9)
1. detect a preparation method for two kinds of acute leukemia mark electrochemical sensors simultaneously, it is characterized in that comprising the following steps:
(1) lysozyme reporter probe and gamma interferon reporter probe are left standstill, with opened disulfide bond respectively in containing the solution of three (2-chloroethyl) phosphate; Lysozyme is added again fit fit with gamma interferon in solution, lysozyme reporter probe and lysozyme fit formation lysozyme reporter probe-fit duplex structure, gamma interferon reporter probe and gamma interferon fit formation gamma interferon reporter probe-fit duplex structure;
(2) pre-service is carried out to gold electrode, the gold electrode handled well is immersed in step (1) pretreated lysozyme reporter probe-fit double-strand and gamma interferon reporter probe-fit double-strand mixed liquor, room temperature hold over night, afterwards with redistilled water and cleaning fluid cleaning;
(3) electrode after step (2) being processed is immersed in the solution containing 6-sulfydryl-1-hexanol, with enclosed-electrode, afterwards with redistilled water and cleaning fluid cleaning;
(4) using step (3) process after electrode as working electrode, and contrast electrode, to Electrode connection on chem workstation, to obtain the electrochemical sensor that can detect two kinds of acute leukemia marks simultaneously;
In step (1): described lysozyme reporter probe is 5 '-sulfydryl-C6-GTGCAGAGCTAAGTAACTCTGCAC-methylene blue-3 ', described lysozyme is fit is 5 '-ATCAGGGCTAAAGAGTGCAGAGTTACTTAG-3 ', described gamma interferon reporter probe is 5 '-sulfydryl-C6-TTCAACCAACTTTTTTTTTTCAACACCCAT-ferrocene-3 ', described gamma interferon is fit is 5 '-GGGGTTGGTTGTGTTGGGTGTTGTGT-3 '.
2. a kind of preparation method simultaneously detecting two kinds of acute leukemia mark electrochemical sensors according to claim 1, it is characterized in that, in step (1): the concentration of described Lysozyme in Solution reporter probe-fit double-strand is 1 μm of ol/L, the concentration of gamma interferon reporter probe-fit double-strand is 1 μm of ol/L; Containing 0.5 μ L10mmol/L tri-(2-chloroethyl) phosphate in described solution, also containing 20mmol/LTris-HCl, 140mmol/LNaCl, 20mmol/LMgCl
2and 20mmol/LKCl, solution ph is 7.4.
3. a kind of preparation method simultaneously detecting two kinds of acute leukemia mark electrochemical sensors according to claim 1, it is characterized in that, in step (1): described standing process is at room temperature carried out, time of repose is 2 hours.
4. according to claim 1ly a kind ofly detect the preparation method of two kinds of acute leukemia mark electrochemical sensors, it is characterized in that, described in step (2), the preprocessing process of gold electrode is as follows simultaneously: the gold electrode Al of 0.3 μm
2o
3powder is polished into minute surface, after redistilled water cleaning, and ultrasonic 50s in redistilled water and ethanolic solution in succession; By the gold electrode cleaned up, in the sulfuric acid solution of 0.5mol/L, cyclic voltammetry scan is to stablizing, and scanning voltage is 0.2 ~ 1.65V, and sweep velocity is 0.1V/s, and then rinse well with redistilled water, room temperature is dried.
5. a kind of preparation method simultaneously detecting two kinds of acute leukemia mark electrochemical sensors according to claim 1, it is characterized in that, in step (3): in described solution, the concentration of MCH is 2mmol/L, also containing 20mmol/LTris-HCl, 140mmol/LNaCl, 20mmol/LMgCl in solution
2and 20mmol/LKCl, solution ph is 7.4.
6. a kind of preparation method simultaneously detecting two kinds of acute leukemia mark electrochemical sensors according to claim 1, it is characterized in that, step (2) is with step (3): described cleaning fluid is the Tris-HCl buffer solution of concentration 10mmol/L, and solution ph is 7.4.
7. the electrochemical sensor prepared by claim arbitrary in claim 1-6 detects while being used for two kinds of acute leukemia mark lysozymes and gamma interferon.
8. detect while electrochemical sensor according to claim 7 is used for two kinds of acute leukemia marks, specifically comprise the following steps:
(1) lysozyme and gamma interferon detect the preparation of solution: lysozyme and gamma interferon are mixed with concentration in the solution and are respectively 0.5 ~ 500nmol/L and 0.5 ~ 500nmol/L, for subsequent use at being stored in 4 DEG C; 20mmol/LTris-HCl, 140mmol/LNaCl, 20mmol/LMgCl is included in described solution
2and 20mmol/LKCl, solution ph is 7.4;
(2) prepared working sensor electrode is immersed in lysozyme that 50 μ L steps (1) prepare and gamma interferon detects in solution, leaves standstill 1h at 37 DEG C, afterwards with redistilled water and cleaning fluid cleaning, obtain the working electrode assembled;
(3) by contrast electrode, be connected on electrochemical workstation to the working electrode of electrode and assembling, the solution of 40 ~ 60 μ L is added in a cell, containing 20mmol/LTris-HCl, 140mmol/LNaCl, 20mmol/LMgCl in solution
2and 20mmol/LKCl, solution ph is 7.4, then detects the current-responsive of the working electrode of assembling with differential pulse voltammetry, according to the relation between gained current-responsive and the concentration of standard solution of two kinds of acute leukemia marks, and drawing curve;
(4) testing sample solution is replaced the standard solution of two kinds of marks, detect according to the method for drafting of the working curve of described two kinds of acute leukemia marks.
9. detect while electrochemical sensor according to claim 8 is used for two kinds of acute leukemia marks, it is characterized in that, in step (2), described cleaning fluid is the Tris-HCl buffer solution of concentration 10mmol/L, and pH value of solution is 7.4.
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| CN106434903B (en) * | 2016-09-07 | 2017-12-19 | 青岛大学 | Detect ratio electrochemical DNA biosensor modified electrode of P53gene and preparation method thereof |
| CN106841340B (en) * | 2017-03-14 | 2019-06-14 | 浙江省农业科学院 | The electrochemical sensor and its method system of isocarbophos and Acetamiprid are detected simultaneously |
| CN106959323B (en) * | 2017-03-20 | 2019-10-01 | 临沂大学 | A method of detection lysozyme |
| CN107290406B (en) * | 2017-06-20 | 2019-05-24 | 中南大学 | For detecting the ferrocene of miRNA and the magnetic ball nano-complex of methylene blue double labeling and preparation method thereof |
| CN108519417B (en) * | 2018-04-16 | 2020-07-03 | 湖南文理学院 | Aptamer probe for detecting two tumor markers, electrochemical biosensor, and preparation method and application of aptamer probe |
| CN110873745A (en) * | 2018-09-04 | 2020-03-10 | 南京大学 | Novel method for detecting acute myelogenous leukemia marker Siglec-5 |
| CN109580745B (en) * | 2018-11-23 | 2021-10-08 | 华中师范大学 | Non-labeled electrochemical biosensor based on aptamer and detection method |
| CN109738500B (en) | 2019-02-20 | 2019-08-06 | 青岛大学 | The preparation method of nano-complex and its label-free aptamer electrochemistry gamma interferon sensor |
| CN111157596A (en) * | 2020-01-03 | 2020-05-15 | 西北大学 | Electrochemical sensor for simultaneously detecting PAT and OTA, preparation method and detection method |
| CN113549623B (en) * | 2021-05-31 | 2023-07-11 | 武汉维尔博生物科技有限公司 | Aptamer combination for simultaneously detecting insulin and glucose, detection sensor and preparation method of detection sensor |
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