CN104597240B - Graphene/class peroxidase leukemic bio-sensing method of dual signal amplification detection - Google Patents
Graphene/class peroxidase leukemic bio-sensing method of dual signal amplification detection Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57426—Specifically defined cancers leukemia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
Abstract
The invention discloses a kind of Graphene/class peroxidase leukemic bio-sensing method of dual signal amplification detection. The invention provides a kind of test kit for leukaemia's detection, containing, for example under (a) and (b): (a) magnetic capture probe; Described magnetic capture probe is made up of magnetic bead and the capture probe being fixed on described magnetic bead; Described capture probe is the single strand dna shown in sequence 1; (b) aptamers functionalization Hemin-graphene composite nano material; Described aptamers functionalization Hemin-graphene composite nano material is made up of with being fixed on described aptamers probe on the Graphene that protohemin is modified the Graphene modified through protohemin; Described aptamers probe is the single strand dna shown in sequence 2. The target leukaemia of low concentration can be caught and be enriched with by the bio-sensing method of the present invention at short notice, amplification detection signal, it is achieved leukaemia is carried out quick, accurate, sensitive detection.
Description
Technical field
The invention belongs to the basic medical application of biosensor, relate to a kind of Graphene/class peroxidase leukemic bio-sensing method of dual signal amplification detection.
Background technology
Leukemia is the Clonal malignant disease of hematopoietic stem cell that a class is common and multiple, is one of big malignant tumor occurred frequently of China ten, occupies the first place of less than 35 years old crowd's mortality of malignant tumors, and its sickness rate has the trend increased year by year. Its diagnosis typing is the FAB classification proposed by method (F), beautiful (A), English (B) cooperative groups for 1976 at first, and it is to diagnose leukemic traditional method. Along with the application in leukemia diagnosis of cytogenetics and immunophenotyping, also been proposed leukemic morphocytology (Morphoiogic), immunology (Immunologic), cytogenetics (Cytogentic) MIC typing. Since the eighties in 20th century, get involved due to MIC sub-type identity sex chromosomal abnormality the clone of gene and continuous anatomy to these gene functions, gradually formed again based on morphocytology, immunology, cytogenetics, molecular biology (Molecular) MIC-M typing.In recent years, along with cytogenetics, the developing rapidly of Protocols in Molecular Biology, the technology such as chromosome analysis, Southern-Blot, RT-PCR and FISH is used to leukemic clinical diagnosis. But these methods still suffer from more shortcoming, such as detection method complexity, radioactive pollution, sensitivity is low, testing expense is expensive, therefore, in clinical diagnosis, the immunological phenotype detection of morphologic detection and Ag-Ab specific recognition is still the basis of leukemia diagnosis. But rely on merely morphological examination to be difficult to carry out classification diagnosis accurately, and immunological phenotype detection needs corresponding antibody and complex operation, expensive, therefore, develop new quick, sensitive, economic leukemia new detecting method and there is very important clinical meaning.
In recent years, nanotechnology develops rapidly and permeates in medical domain, and the early diagnosis for tumor brings dawn. NIH started " cancer nanotechnology plan " in 2005, it is intended to nanotechnology, cancer research and molecular biosciences medical science are be combined with each other, it is achieved alleviate the painful target with reduction mortality rate of cancer patient. Graphene (graphene) is a kind of carbon two-dimensional nanostructure consisted of sp2 hydridization, is the essential structure unit of every other dimension (such as zero dimension fullerene, one-dimensional CNT, three-dimensional graphite etc.) carbonaceous material. It has nanostructured and heat, machinery and electrical properties, the advantage such as big in specific surface area, surface reaction activity is high, catalytic efficiency is high, high adsorption capacity of uniqueness, is particularly well-suited to sensing and the amplification of fixing, the signal of sensitive molecule. Haemachrome is a kind of iron porphyrin coordination compound, the core of internal hemoglobin of behaving, owing to it is cheap, stable in properties and can the reduction of catalytic oxygen, hydrogen peroxide etc. and be widely used, but its catalysis activity and electrochemical stability unsatisfactory. By Hemin functionalization graphene nano-hybrid material (HGNs) of π-π interaction preparation between Hemin and Graphene, there is good stability in aqueous, the haemachrome of graphenic surface attachment makes HGNs have catalatic character, and Graphene can improve the catalysis Activity and stabill of Hemin. Therefore, HGNs can as a kind of Application of micron with Catalyzed Synthesis By Peroxidase activity in the preparation of biosensor, and HGNs is applied to the detection of tumor cell, and there is not been reported.
Summary of the invention
It is an object of the invention to provide a kind of test kit for leukaemia's detection.
Test kit for leukaemia's detection provided by the present invention, containing, for example under (a) and (b):
(a) magnetic capture probe;
Described magnetic capture probe is made up of magnetic bead and the capture probe being fixed on described magnetic bead; Described capture probe is the single strand dna shown in sequence 1 in sequence table;
(b) aptamers functionalization Hemin-graphene composite nano material;
Described aptamers functionalization Hemin-graphene composite nano material is made up of with being fixed on described aptamers probe on the Graphene that protohemin is modified the Graphene modified through protohemin (Hemin); Described aptamers probe is the single strand dna shown in sequence 2 in sequence table.
In described test kit, described aptamers functionalization Hemin-graphene composite nano material specifically can be prepared by a method comprising the following steps: according to the ratio that proportioning is 0.5mg:4-40nmol (such as 1mg:8nmol) of the described Graphene through protohemin modification and described aptamers probe, the described suspension of Graphene modified through protohemin is mixed with the solution of described aptamers probe, 20-25 DEG C of (such as 25 DEG C) lucifuge hatches 16-24h (such as 24h), obtain described aptamers functionalization Hemin-graphene composite nano material.
Wherein, described in the suspension of the Graphene of protohemin modification, the described content through the Graphene of protohemin modification is 1mg/ml. In the solution of described aptamers probe, the concentration of described aptamers probe is 8 μMs.
In the period carrying out described 20-25 DEG C of lucifuge and hatching 16-24h (such as 24h), also include addition NaCl in incubation system, to the step of the final concentration of 10mM of described NaCl.
Wherein, the described NaCl that adds in incubation system for gradually to add NaCl in described incubation system; Particularly as follows: each 0.5 μ LNaCl solution (concentration is 1M), within every 5 hours, add once.
In described test kit, the described Graphene modified through protohemin is to be 1:(1～10 by graphene oxide and protohemin according to mass ratio) ratio of (such as 1:1) prepares.
The described Graphene modified through protohemin is specifically prepared by a method comprising the following steps: be 1:(1～10 by graphene oxide and protohemin according to mass ratio) after the ratio mixing of (such as 1:1), add ammonia and hydrazine hydrate, hatch 2-6h (such as 4h) for 60 DEG C, obtain the described Graphene modified through protohemin.
Wherein, described addition ammonia and hydrazine hydrate particularly as follows: the ratio of the described ammonia (mass fraction is 25-28%) that adds and protohemin is 10 μ L:1mg, the described hydrazine hydrate (mass fraction is 80%) of addition and the ratio 3 μ L:1mg of graphene oxide.
In described test kit, described magnetic capture probe is prepared by a method comprising the following steps: jointly will hatch with the described magnetic bead through marked by streptavidin through biotin labeled described capture probe, it is thus achieved that described magnetic capture probe.
In the present invention, described biotin is specifically marked on 3 ' ends of described capture probe.
When hatching described in carrying out, it is specially 100pmol:1mg through biotin labeled described capture probe with the proportioning of described magnetic bead through marked by streptavidin;
Further, hatch described in and be specially 20-30 DEG C of (such as 25 DEG C) 80-150rpm (such as 100rpm) concussion and hatch 10-30min (such as 15min). The described liquid environment hatched is binding buffer liquid; The solvent of described binding buffer liquid is that water, solute and concentration are as follows: Tris0.605g/L, EDTA0.185g/L, NaCl58.45g/L, HCl regulate pH to 7.5.
More concrete, in one embodiment of the invention, the preparation method of described magnetic capture probe comprises the steps: to take the solution through biotin labeled described capture probe (solvent is for described binding buffer liquid) that concentration is 500nM (measuring with DNA) and the described magnetic bead solution through marked by streptavidin (solvent is for described binding buffer liquid) that concentration is 10mg/ml (measuring with magnetic bead) respectively, mix according to the proportioning of volume ratio 2:1, 10-30min (such as 15min) is hatched in 20-30 DEG C of (such as 25 DEG C) 80～150rpm (such as 100rpm) concussion, namely described magnetic capture probe is obtained.
In described test kit, also can contain the readable carrier (such as description) recording following (A) and/or (B):
(A) detection or auxiliary detect the method whether cell to be measured is leukaemia, comprise the steps:
(a1) by described aptamers functionalization Hemin-graphene composite nano material, cell to be measured and described magnetic capture probe according to 25 μ g:105Individual: the proportioning mixing of 25 μ g, 37 DEG C of lucifuges hatch 30min;The amount of described magnetic capture probe is with magnetic bead gauge;
As required, may also include, described hatching, the step adopting cleaning buffer solution that product is carried out afterwards; The solvent of described cleaning buffer solution is that water, solute and concentration are as follows: NaCl8g/L, KCl0.2g/L, CaCl20.14g/L, Na2HPO4·H2O0.1g/L, MgCl2·6H2O1.42g/L, NaHCO30.35g/L, KH2PO40.2g/L, glucose 4.5g/L.
(a2) system after step (a1) being hatched carries out magnetic enrichment, removes supernatant, adds TMB nitrite ion and hydrogen peroxide in enriched product, and 37 DEG C of lucifuges hatch 30min;
(a3) determine whether cell to be measured is leukaemia as follows: if step (a2) hatch after system relatively hatch before compare and there occurs chromogenic reaction (namely reaction system is become blue by colourless), then described cell to be measured is or candidate is leukaemia; Otherwise, then described cell to be measured is not or candidate is for leukaemia;
(B) detect the method for leukaemia's content in cell sample to be measured, comprise the steps:
(B1) acquisition of standard curve:
(b1) suspensions different to described aptamers functionalization Hemin-graphene composite nano material, series standard leukaemia's content and described magnetic capture probe are mixed according to the proportioning of 25 μ g:20 μ l:25 μ g, obtain the standards system to be measured that some parts described standard leukemia cell content is known, hatch 30min in 37 DEG C of lucifuges; The amount of described magnetic capture probe is with magnetic bead gauge;
Wherein, by described aptamers functionalization Hemin-graphene composite nano material, suspension that series standard leukaemia's content is different and described magnetic capture probe mix particularly as follows: by the suspension (liquid is described cleaning buffer solution) of described aptamers functionalization Hemin-graphene composite nano material that described aptamers functionalization Hemin-graphene composite nano material content is 0.5mg/ml according to the proportioning of 25 μ g:20 μ l:25 μ g, the suspension (liquid is described cleaning buffer solution) that described series standard leukaemia's content is different, with the solution (solvent is described cleaning buffer solution) of the described magnetic capture probe that concentration is 10mg/ml (with magnetic bead gauge) according to the ratio mixing that volume ratio is 25:20:2.5. in the suspension that series standard leukaemia's content described in every 20 μ l is different, the content of described standard leukemia cell is followed successively by 105Individual, 104Individual, 103Individual, 102Individual, 101Individual, 0.
As required, may also include, described hatching, the step adopting cleaning buffer solution that product is carried out afterwards; The solvent of described cleaning buffer solution is that water, solute and concentration are as follows: NaCl8g/L, KCl0.2g/L, CaCl20.14g/L, Na2HPO4·H2O0.1g/L, MgCl2·6H2O1.42g/L, NaHCO30.35g/L, KH2PO40.2g/L, glucose 4.5g/L.
(b2) the some systems after step (b1) being hatched carry out magnetic enrichment, remove supernatant, add TMB nitrite ion and hydrogen peroxide in enriched product, and 37 DEG C of lucifuges hatch 30min;
(b3) the some systems after step (b2) being hatched carry out ultraviolet absorption value detection, and detection wavelength is 550-800nm (such as 650nm);
(b4) logarithm of the standard leukemia cell content of the ultraviolet absorption value of the described some systems obtained according to step (b3), and correspondence, it is thus achieved that the standard curve equation between logarithm and the ultraviolet absorption value of standard leukemia cell content;
Wherein, described standard leukemia cell is specially cem cell (human T lymphocyte leukaemia).
(B2) the standard curve equation adopting step (B1) gained measures the content of leukaemia in cell sample to be measured according to the method comprising the steps (b5)-(b6):
(b5) being operated according to step (b1)-(b3), (in cell sample suspension to be measured described in 20 μ l, total cellular score is as 10 only suspensions different for described series standard leukaemia's content to be replaced with cell sample suspension to be measured5Individual), all the other operations are all identical;
(b6) ultraviolet absorption value step (b5) recorded substitutes in the standard curve equation that step (B1) obtains, thus obtaining the content of leukaemia in described cell sample to be measured.
In step (a2) and (b2), described TMB nitrite ion and the hydrogen peroxide of adding in enriched product particularly as follows: add TMB nitrite ion described in 100 μ l (as formula is: solvent is DMSO, TMB concentration is 0.6mg/ml) and 1 μ l500mM hydrogen peroxide in enriched product.
Described Hemin-graphene composite nano material falls within protection scope of the present invention.
The application in the described test kit detected for leukaemia of preparation of the described Hemin-graphene composite nano material falls within protection scope of the present invention.
Described leukaemia can be cem cell.
Test kit provided by the present invention is based on Graphene/class peroxidase leukemic bio-sensing method of dual signal amplification detection, there is the advantage of following uniqueness: (1) utilizes the feature of aptamer high-affinity and high specific, and magnetic catches the dual signal amplification with Graphene, effectively raise susceptiveness and the specificity of the method; (2) using aptamer as identifying probe, more stable compared to the antibody in traditional method and economical; (3) detection signal naked eyes are visible, it is not necessary to expensive instrument and complicated operation; (4) the detection time is short, just can complete in one hour, more economical and convenient compared to traditional method. To sum up, the target leukaemia of low concentration can be caught and be enriched with by the bio-sensing method of the present invention at short notice, amplification detection signal, it is achieved leukaemia is carried out quick, accurate, sensitive detection.
Accompanying drawing explanation
Fig. 1 is the characteristic absorption peak through the Hemin Graphene modified and electrochemical Characterization figure. A is characteristic absorption peak testing result; B is electrochemical Characterization figure.
Fig. 2 is the principle simulation figure of the Graphene/class peroxidase leukemic bio-sensing method of dual signal amplification detection of the present invention.
Fig. 3 is the bio-sensing method of the Graphene/class peroxidase dual signal amplification detection leukaemia standard curve to different number cem cell detections.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
Cem cell (human T lymphocyte leukaemia): purchased from bio tech ltd of upper Haifeng county longevity, article No.: Cell-3720.
Ramos cell (human B lymphocyte oncocyte): purchased from bio tech ltd of upper Haifeng county longevity, article No.: Cell-0796.
Graphene oxide: Nanjing Xian Feng Nono-material Science & Technology Ltd. product, its catalog number is XF002.
Protohemin (Hemin): sigma Products, its catalog number is 51280 (please indicate).
Embodiment 1, the preparation of test kit for leukaemia's detection
For the test kit of leukaemia's detection contains magnetic capture probe and aptamers functionalization Hemin-graphene composite nano material.
One, the preparation of magnetic capture probe
(1) through the preparation of biotin labeled capture probe
Described biotin labeled capture probe is biotin labeled single strand dna, and for the raw work Products of Chinese Shanghai, the single-stranded DNA sequence used by this probe is as follows:
5 '-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGAAAAAA-3 ' (sequence 1), wherein biotin labeling molecule is at 3 ' ends of nucleic acid chains.
(2) through the magnetic bead of marked by streptavidin
The magnetic bead of described marked by streptavidin be Invitrogen company produce Dynabeads Streptavidin MagneSphere M280, the design parameter of this magnetic bead is as follows: as magnetic bead average diameter 2.8 μm, surface area: 4-8m2/g, density: 1.4g/cm3 (I does not find), magnetic bead content: 10mg/ml, isoelectric point, IP: pH5.0, low electric charge :-10mV (pH7) (I does not find), CV value < 3%, iron content (ferrite): 12% (17%).
(3) preparation of magnetic capture probe
1, it is configured to, through biotin labeled capture probe binding buffer liquid, the solution that concentration is 500nM (measuring with DNA) by what step () obtained. Wherein, the solvent of described binding buffer liquid is that water, solute and concentration are as follows: Tris0.605g/L, EDTA0.185g/L, NaCl58.45g/L, HCl regulate pH to 7.5.
2, (concentration is 10mg/ml to take 200 μ l step (two) magnetic bead through marked by streptavidin obtained, calculate with the gauge of magnetic bead), after washing 2 times with binding buffer liquid (formula is ibid), then magnetic bead is suspended in the binding buffer liquid of 200 μ l.
3, (concentration is for 10mg/ml to take the magnetic bead of the marked by streptavidin processed through step 2 through biotin labeled capture probe solution and 200 μ l that the concentration that 400 μ l steps 1 obtain is 500nM (measuring with DNA), calculate with the gauge of magnetic bead) fully after mixing, 15 minutes are hatched in 25 DEG C of 100rpm concussions, put on magnetic frame, after Magneto separate, abandoning supernatant, gained precipitation binding buffer liquid (formula is ibid) is resuspended.
4, clean twice with the binding buffer liquid (formula is ibid) of 600 μ l, finally with the resuspended magnetic bead being fixed with DNA of 200 μ l binding buffer liquid (formula is ibid) to 10mg/ml (in magnetic bead).
Two, the preparation of Hemin-graphene composite nano material
1, through the preparation of the Hemin Graphene modified and qualification
(1) through the preparation of the Hemin Graphene modified
After the graphene oxide suspension (liquid is water) that 1mL concentration is 1mg/mL is mixed with the protohemin (Hemin) (solvent is DMSO) that 1mL concentration is 1mg/mL, in mixed system, (ammonia (mass fraction 25-28%) of addition is 10 μ L for addition ammonia and hydrazine hydrate, hydrazine hydrate (mass fraction 80%) is 3 μ L), in 60 DEG C of water-baths 4 hours, lyophilisation prepared through the Hemin Graphene (being designated as HGNs) modified.
(2) through the qualification of the Hemin Graphene modified
On the one hand, carrying out uv absorption blob detection to through the Hemin Graphene modified, concrete operations are as follows: being added in cuvette by the 100 μ LHemin Graphene suspension modified, with ultraviolet spectrophotometer, it is detected, wave-length coverage is 200-600nm. Experiment arranges graphene oxide, protohemin (Hemin) as comparison simultaneously.
On the other hand, carrying out electrochemical signals detection to through the Hemin Graphene modified, concrete operations are as follows: on glass-carbon electrode, 25 DEG C Hemin Graphene (concentration is 0.5mg/ml) the 20 μ L modified dropping is being hatched 12h.At PBS, (solvent is that water, solute and concentration are as follows: NaCl8g/L, KCl2g/L, Na2HPO4·H2O3.5g/L, KH2PO40.2g/L) signal of Hemin is detected (using saturated calomel electrode as reference electrode) by middle differential pulse voltametry (DPV), and potential range is: 0～-0.6V. Experiment arranges the glass-carbon electrode of naked glass-carbon electrode and modification graphene oxide as comparison simultaneously.
In characteristic absorption peak testing result such as Fig. 1 shown in A, the characteristic absorption peak of graphene oxide is at about 233nm, the characteristic absorption peak of Hemin is at about 384nm, by reducing, Hemin is loaded to after on Graphene, this occurs in 264nm and 413nm through the absworption peak of the Hemin Graphene (HGNs) modified, all there is red shift in the absworption peak compared to graphene oxide and Hemin, illustrates that Hemin successfully loads to graphenic surface.
In electrochemical signals testing result such as Fig. 1 shown in B, all there is not obvious electrochemical signals in the glass-carbon electrode of naked glass-carbon electrode and modification graphene oxide, and having modified the electrochemical signals demonstrating Hemin through the Hemin glass-carbon electrode of Graphene (HGNs) modified, this electrochemistry collection of illustrative plates display Hemin successfully loads to graphenic surface.
2, the preparation of aptamers functionalization Hemin-graphene composite nano material
Aptamers probe:
5 '-GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAATCTAACTGCTGCGCCGCCGGGAAAA TACTGTACGGTTAGA-3 ' (sequence 2)
(liquid is binding buffer liquid to the Graphene suspension (liquid is water) through the Hemin modification step 1 that 100 μ L concentration are 1mg/mL prepared with the aptamers probe solution that 100 μ L concentration are 8 μMs, (for 1L solution) composed as follows: Tris0.605g/L, EDTA0.185g/L, NaCl58.45g/L, HCl regulates pH to 7.5) after mix homogeneously, room temperature (25 DEG C) lucifuge hatches 24 hours, period gradually adds aging (each 0.5 μ LNaCl aqueous solution (concentration is 1M) of aqueous solution (final concentration of 10mM) of NaCl, within every 5 hours, add once, totally 4 times), product is the Hemin-graphene composite nano material of aptamers probe functionalization.
Three, for the using method of the test kit of leukaemia's detection
Test kit provided by the present invention is based on the leukemic bio-sensing method of Graphene/class peroxidase dual signal amplification detection and realizes detection, and its principle simulation figure is as shown in Figure 2. Specifically include herein below:
The Hemin-graphene composite nano material of leukaemia's aptamers probe functionalization can specific identification in conjunction with leukaemia; Magnetic capture probe energy specific enrichment leukaemia. When target cell exists, its Hemin-graphene composite nano material with aptamers probe functionalization and magnetic capture probe form sandwich structure, under the influence of a magnetic field, this sandwich structure is attracted near Magnet, now containing substantial amounts of Graphene/class peroxidase composite nano materials in this sandwich structure, add substrate TMB, chromogenic reaction can occur, utilize ultraviolet-uisible spectrophotometer that it is detected, obvious uv absorption signal can be obtained. When target cell is absent from, being adsorbed near Magnet is magnetic capture probe, a large amount of Graphenes/class peroxidase composite nano materials is free in supernatant and is removed, add substrate TMB, chromogenic reaction can not be there is, by the detection of ultraviolet-uisible spectrophotometer, it is only capable of obtaining faint uv absorption signal or no signal. Observe whether system chromogenic reaction occurs by adding substrate TMB, it may be achieved the qualitative detection to leukaemia;Utilize the change of uv absorption signal, it may be achieved the quantitative analysis to leukaemia; By replacing different capture probes, the method can also realize the detection to other biological mark, cell, metal ion etc., make a kind of general bioanalytical sensing platform, detection for leukaemia provides new approaches, follows the tracks of the new technique, the new method that provide economical, convenient, practical for the major disease early warnings such as tumor, early diagnosis and curative effect.
1, the qualitative checking method of test kit of the present invention
Adopt this test kit to detect whether cell to be measured is leukaemia, specifically include following steps:
(1) (liquid is cleaning buffer solution, and solvent is that water, solute and concentration are as follows: NaCl8g/L, KCl0.2g/L, CaCl to take the aptamers functionalization Hemin-graphene composite nano material suspension that step 2 that 50 μ l concentration are 0.5mg/ml prepares20.14g/L, Na2HPO4·H2O0.1g/L, MgCl2·6H2O1.42g/L, NaHCO30.35g/L, KH2PO40.2g/L, glucose 4.5g/L), 20 μ l cells (10 to be measured5Individual cell, liquid is cleaning buffer solution) and 2.5 μ l concentration be magnetic capture probe suspension (liquid is for the cleaning buffer solution) mix homogeneously that 10mg/ml (with magnetic bead gauge) step one prepares, 37 DEG C of lucifuges hatch 30min;
(2) product is washed twice with cleaning buffer solution (formula is ibid), magnetic enrichment is carried out after washing, remove supernatant, 100 μ lTMB nitrite ions (formula: solvent is DMSO is added in enriched product, TMB concentration is 0.6mg/ml) and 1 μ l500mM hydrogen peroxide mix homogeneously, 37 DEG C of lucifuges hatch 30min;
(3) determine whether cell to be measured is leukaemia as follows: if step (2) hatch after system relatively hatch before compare and there occurs chromogenic reaction (namely reaction system is become blue by colourless), then described cell to be measured is leukaemia; Otherwise, then described cell to be measured is not leukaemia.
2, the quantitative detecting method of test kit of the present invention
Adopt this test kit to detect leukaemia's content in cell sample to be measured, specifically include following steps:
A. the acquisition of standard curve
(1) take the Hemin-graphene composite nano material suspension (liquid is described cleaning buffer solution) of aptamers functionalization that step 2 that 50 μ l concentration are 0.5mg/ml prepares, 20 μ lCEM cell samples (arrange number of cells for such as Gradient: 105Individual, 104Individual, 103Individual, 102Individual, 101Individual, 0, liquid is described cleaning buffer solution) and 2.5 μ l concentration be 10mg/ml (with magnetic bead gauge) step one prepare magnetic capture probe suspension (liquid is described cleaning buffer solution) mix homogeneously, 37 DEG C of lucifuges hatch 30min;
(2) product is washed twice with cleaning buffer solution (formula is ibid), magnetic enrichment is carried out after washing, remove supernatant, 100 μ lTMB nitrite ions (formula: solvent is DMSO is added in enriched product, TMB concentration is 0.6mg/ml) and 1 μ l500mM hydrogen peroxide mix homogeneously, 37 DEG C of lucifuges hatch 30min;
(3) with ultra-violet and visible spectrophotometer, each sample being carried out ultraviolet absorption value detection, detection wavelength is 650nm;
(4) ultraviolet absorption value (OD650) of each sample obtained according to step (3), and the logarithm of the quantity of the cem cell of correspondence (lg), obtain the standard curve equation between logarithm (lg) and the ultraviolet absorption value (OD650) of cem cell quantity, abscissa (x) represents the logarithm (lg) of cem cell quantity, and vertical coordinate (y) represents ultraviolet absorption value (OD650);
B. the standard curve equation adopting step A gained measures the content of leukaemia in cell sample to be measured according to the method comprised the steps:
(5) being operated according to (1) in step A-(3), (total cellular score is 10 only 20 μ lCEM cell samples to be replaced with 20 μ l cell samples to be measured5Individual);
(6) OD650 recorded is substituted into the standard curve equation of step A gained, obtains the logarithm of leukaemia's quantity in 20 μ l cell samples to be measured thus calculating, and then obtain the content of leukaemia in cell sample to be measured.
Embodiment 2, the application example of test kit for leukaemia's detection
One, the test kit adopting embodiment 1 detects whether cell to be measured is leukaemia
Cell sample to be measured: cem cell (people's Pancytopenia cell), Ramos cell (human B lymphocyte oncocyte).
Detecting as cell sample to be measured with reference to the method recorded in embodiment 1 step 3 A, experiment arranges Ramos cell (human B lymphocyte oncocyte) simultaneously and is negative control group, does not add the blank group of any cell.
Result shows, cem cell group has obvious chromogenic reaction (namely reaction system is become blue by colourless), and other groups are then the same with blank group, it does not have chromogenic reaction produces. Should it is shown that test kit provided by the present invention can realize the specific detection to leukaemia.
Two, the test kit adopting embodiment 1 detects the content of leukaemia in cell sample to be measured
Cell sample to be measured: containing 2 × 103Suspension (liquid the is described cleaning buffer solution) sample of individual cem cell.
Detecting as cell sample to be measured with reference to the method recorded in embodiment 1 step 3 B, experiment arranges the blank group not adding any cell simultaneously.
Result shows:
1, the standard curve equation between logarithm (lg) and the ultraviolet absorption value (OD650) of cem cell quantity is y=0.016x+0.0419 (R2=0.9759), specifically as shown in Figure 3. As seen from the figure, ultraviolet absorption value (OD650) increases with analyte cem cell number and increases, 101～105With light absorption value linearly, Monitoring lower-cut is up to 10 cells for the logarithm value of individual cell context inner cell number.
2, the OD650 of measured cell sample to be measured is 0.095, substitute into standard curve equation, the logarithm (lg) obtaining cem cell quantity is 3.3187, and then calculates that to obtain cem cell quantity quantity in cell sample to be measured be 2083, is consistent with actual. Visible, test kit provided by the present invention can realize the detection by quantitative to leukaemia, and testing result is accurately and reliably.
Claims (12)
1. for a test kit for leukaemia's detection, containing, for example under (a) and (b):
(a) magnetic capture probe;
Described magnetic capture probe is made up of magnetic bead and the capture probe being fixed on described magnetic bead; Described capture probe is the single strand dna shown in sequence 1 in sequence table;
(b) aptamers functionalization Hemin-graphene composite nano material;
Described aptamers functionalization Hemin-graphene composite nano material is made up of with being fixed on described aptamers probe on the Graphene that protohemin is modified the Graphene modified through protohemin; Described aptamers probe is the single strand dna shown in sequence 2 in sequence table.
2. test kit according to claim 1, it is characterized in that: described aptamers functionalization Hemin-graphene composite nano material is prepared by a method comprising the following steps: according to the ratio that proportioning is 0.5mg:4-40nmol of the described Graphene through protohemin modification and described aptamers probe, the described suspension of Graphene modified through protohemin is mixed with the solution of described aptamers probe, 20-25 DEG C of lucifuge hatches 16-24h, obtains described aptamers functionalization Hemin-graphene composite nano material.
3. test kit according to claim 2, it is characterized in that: in the period carrying out described 20-25 DEG C of lucifuge and hatching 16-24h, also include in incubation system, add NaCl and obtain containing NaCl system, NaCl described be the step of 10mM containing the concentration in NaCl system.
4. according to described test kit arbitrary in claim 1-3, it is characterised in that: the described Graphene modified through protohemin is to be 1:(1～10 by graphene oxide and protohemin according to mass ratio) ratio prepare.
5. test kit according to claim 4, it is characterized in that: the described Graphene modified through protohemin is prepared by a method comprising the following steps: be 1:(1～10 by graphene oxide and protohemin according to mass ratio) ratio mixing after, add ammonia and hydrazine hydrate, hatch 2～6h for 60 DEG C, obtain the described Graphene modified through protohemin.
6. according to described test kit arbitrary in claim 1-3, it is characterized in that: described magnetic capture probe is prepared by a method comprising the following steps: jointly will hatch with the described magnetic bead through marked by streptavidin through biotin labeled described capture probe, it is thus achieved that described magnetic capture probe.
7. test kit according to claim 6, it is characterised in that: when hatching described in carrying out, it is 100pmol:1mg through biotin labeled described capture probe with the proportioning of described magnetic bead through marked by streptavidin.
8. test kit according to claim 6, it is characterised in that: described in hatch and hatch 10-30min for 20-30 DEG C of concussion.
9. test kit according to claim 6, it is characterised in that: described in hatch and carry out in binding buffer liquid; The solvent of described binding buffer liquid is that water, solute and concentration are as follows: Tris0.605g/L, EDTA0.185g/L, NaCl58.45g/L; The pH value of described binding buffer liquid is 7.5.
10. according to described test kit arbitrary in claim 1-3, it is characterised in that: possibly together with the readable carrier recording following (A) and/or (B) in described test kit:
(A) detection or auxiliary detect the method whether cell to be measured is leukaemia, comprise the steps:
(a1) by described aptamers functionalization Hemin-graphene composite nano material, cell to be measured and described magnetic capture probe according to 25 μ g:105Individual: the proportioning mixing of 25 μ g, 37 DEG C of lucifuges hatch 30min; The amount of described magnetic capture probe is with magnetic bead gauge;
(a2) system after step (a1) being hatched carries out magnetic enrichment, obtains enriched product, adds TMB and hydrogen peroxide in described enriched product, and 37 DEG C of lucifuges hatch 30min;
(a3) determine whether cell to be measured is leukaemia as follows: if step (a2) hatch after system relatively hatch before compare and there occurs chromogenic reaction, then described cell to be measured is or candidate is leukaemia; Otherwise, then described cell to be measured is not or candidate is for leukaemia;
(B) detect the method for leukaemia's content in cell sample to be measured, comprise the steps:
(B1) acquisition of standard curve:
(b1) suspensions different to described aptamers functionalization Hemin-graphene composite nano material, series standard leukaemia's content and described magnetic capture probe are mixed according to the proportioning of 25 μ g:20 μ l:25 μ g, obtain the standards system to be measured that some parts described standard leukemia cell content is known, hatch 30min in 37 DEG C of lucifuges;The amount of described magnetic capture probe is with magnetic bead gauge;
(b2) the some systems after step (b1) being hatched carry out magnetic enrichment, obtain enriched product, add TMB nitrite ion and hydrogen peroxide in enriched product, and 37 DEG C of lucifuges hatch 30min;
(b3) the some systems after step (b2) being hatched carry out ultraviolet absorption value detection, and detection wavelength is 550-800nm;
(b4) ultraviolet absorption value of the described some systems obtained according to step (b3), and the logarithm of the described standard leukemia cell content of correspondence, it is thus achieved that the standard curve equation between logarithm and the described ultraviolet absorption value of described standard leukemia cell content;
(B2) the standard curve equation adopting step (B1) gained measures the content of leukaemia in cell sample to be measured according to the method comprising the steps (b5)-(b6):
(b5) being operated according to step (b1)-(b3), only suspensions different for described series standard leukaemia's content is replaced with cell sample suspension to be measured, all the other operations are all identical;
(b6) ultraviolet absorption value step (b5) recorded substitutes in the standard curve equation that step (B1) obtains, thus obtaining the content of leukaemia in described cell sample to be measured.
The aptamers functionalization Hemin-graphene composite nano material described in 11. claim 1-5 is arbitrary.
12. the aptamers functionalization Hemin-graphene composite nano material described in claim 9 is the application in arbitrary described test kit being used for leukaemia's detection in preparation claim 1-10.
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