CN104597240A - Biosensing method for detecting leukemia by graphene/mimetic peroxidase double-signal amplification - Google Patents
Biosensing method for detecting leukemia by graphene/mimetic peroxidase double-signal amplification Download PDFInfo
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- CN104597240A CN104597240A CN201510053919.1A CN201510053919A CN104597240A CN 104597240 A CN104597240 A CN 104597240A CN 201510053919 A CN201510053919 A CN 201510053919A CN 104597240 A CN104597240 A CN 104597240A
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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 biosensing method for detecting leukemia by graphene/mimetic peroxidase double-signal amplification. The invention provides a kit for detecting leukemia cells; the kit comprises (a) a magnetic bead capture probe and (b) an aptamer functionalization Hemin-graphene composite nanomaterial, wherein the magnetic bead capture probe comprises a magnetic bead and a capture probe fixed on the magnetic bead; the capture probe is a single-stranded deoxyribonucleic acid (DNA) molecule shown by sequence 1; the aptamer functionalization Hemin-graphene composite nanomaterial is composed of graphene modified by Hemin, and an aptamer probe fixed on the graphene modified by the Hemin; the aptamer probe is a single-stranded DNA molecule shown by sequence 2. After the biosensing method is used, the target leukemia cells with lower concentration can be captured and enriched within a short time, and a detection signal is amplified, so that the leukemia cells can be rapidly, accurately and sensitively detected.
Description
Technical field
The invention belongs to the basic medical application of biology sensor, relate to a kind of Graphene/leukemic bio-sensing method of class peroxidase dual signal amplification detection.
Background technology
Leukaemia is the common and multiple Clonal malignant disease of candidate stem cell of a class, and be one of large malignant tumour occurred frequently of China ten, occupy the first place of less than 35 years old crowd's mortality of malignant tumors, its incidence of disease has the trend increased year by year.Its diagnosis typing is that the FAB proposed by method (F), beautiful (A), English (B) cooperative groups for 1976 classifies at first, and it is the leukemic classic method of diagnosis.Along with the application in leukemia diagnosis of cytogenetics and immunophenotyping, also been proposed leukemic cytomorphology (Morphoiogic), immunology (Immunologic), cytogenetics (Cytogentic) MIC somatotype.Since the eighties in 20th century, because MIC sub-type identity sex chromosomal abnormality is got involved the clone of gene and the continuous anatomy to these gene functions, define again the MIC-M somatotype based on cytomorphology, immunology, cytogenetics, molecular biology (Molecular) gradually.In recent years, along with the develop rapidly of cytogenetics, Protocols in Molecular Biology, the technology such as chromosome analysis, Southern-Blot, RT-PCR and FISH is used to leukemic clinical diagnosis.But still there is more shortcoming in these methods, as detection method complexity, radioactive contamination, sensitivity is low, testing expense is expensive, therefore, in clinical diagnosis, the immunological phenotype of morphologic detection and Ag-Ab specific recognition detects the basis being still leukemia diagnosis.But simple dependence morphological examination is difficult to carry out classification diagnosis accurately, and immunological phenotype detects the corresponding antibody of needs, and complex operation, expensive, therefore, develop new quick, sensitive, economic leukaemia new detecting method and there is very important clinical meaning.
In recent years, nanometer technology develops rapidly and permeates in medical domain, for the early diagnosis of tumour brings dawn.NIH started in 2005 " plan of cancer nanometer technology ", was intended to nanometer technology, cancer research and molecular biosciences medical science to be combined with each other, and realized alleviating the painful target with reducing mortality ratio 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 (as zero dimension fullerene, one dimension carbon nano-tube, three-dimensional graphite etc.) carbonaceous material.It has unique nanostructured and heat, machinery and electrical properties, the advantages such as large in specific surface area, surface reaction activity is high, catalytic efficiency is high, high adsorption capacity, is specially adapted to sensing and the amplification of fixing, the signal of sensitive molecule.Protoheme is a kind of iron porphyrin complex, is the core of haemoglobin in human body, because it is cheap, stable in properties and can catalytic oxygen, hydrogen peroxide etc. reduction and be widely used, but its catalytic activity and electrochemical stability unsatisfactory.By the Hemin functionalization graphene nano-hybrid material (HGNs) of π-π interaction preparation between Hemin and Graphene, there is good stability in aqueous, the protoheme of graphenic surface attachment makes HGNs have catalatic character, and Graphene can improve catalytic activity and the stability of Hemin.Therefore, HGNs can be used as a kind of Application of micron with Catalyzed Synthesis By Peroxidase activity in the preparation of biology sensor, and there is not been reported HGNs to be applied to the detection of tumour cell.
Summary of the invention
The object of this invention is to provide a kind of kit detected for leukaemia.
The kit detected for leukaemia 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 be fixed on described magnetic bead; Described capture probe is the single strand dna shown in sequence in sequence table 1;
(b) aptamers functionalization Hemin-graphene composite nano material;
Described aptamers functionalization Hemin-graphene composite nano material by the Graphene modified through X-factor (Hemin) and be fixed on described through X-factor modify Graphene on aptamers probe form; Described aptamers probe is the single strand dna shown in sequence in sequence table 2.
In described kit, described aptamers functionalization Hemin-graphene composite nano material specifically can prepare according to the method comprised the steps: the ratio according to the described Graphene modified through X-factor and the proportioning of described aptamers probe being 0.5mg:4-40nmol (as 1mg:8nmol), the suspension of the described Graphene through X-factor modification is mixed with the solution of described aptamers probe, 20-25 DEG C of (as 25 DEG C) lucifuge hatches 16-24h (as 24h), obtain described aptamers functionalization Hemin-graphene composite nano material.
Wherein, in the suspension of the described Graphene through X-factor modification, the content of the described Graphene through X-factor modification is 1mg/ml.In the solution of described aptamers probe, the concentration of described aptamers probe is 8 μMs.
Carrying out during a described 20-25 DEG C lucifuge hatches 16-24h (as 24h), also comprise and add NaCl in incubation system, the final concentration to described NaCl is the step of 10mM.
Wherein, describedly in incubation system, NaCl is added for successively to add NaCl in described incubation system; Be specially: each 0.5 μ L NaCl solution (concentration is 1M), adds once for every 5 hours.
In described kit, the described Graphene modified through X-factor is 1:(1 ~ 10 by graphene oxide and X-factor according to mass ratio) ratio of (as 1:1) prepares.
The described Graphene modified through X-factor specifically prepares according to the method comprised the steps: be 1:(1 ~ 10 by graphene oxide and X-factor according to mass ratio) after the ratio mixing of (as 1:1), add ammoniacal liquor and hydrazine hydrate, hatch 2-6h (as 4h) for 60 DEG C, obtain the described Graphene modified through X-factor.
Wherein, describedly add ammoniacal liquor and hydrazine hydrate is specially: the described ammoniacal liquor (massfraction is 25-28%) added is 10 μ L:1mg with the ratio of X-factor, the ratio 3 μ L:1mg of the described hydrazine hydrate (massfraction is 80%) added and graphene oxide.
In described kit, described magnetic capture probe prepares according to the method comprised the steps: jointly hatch through biotin labeled described capture probe and the described magnetic bead through marked by streptavidin, obtain described magnetic capture probe.
In the present invention, described biotin is specifically marked on 3 ' end of described capture probe.
When hatching described in carrying out, be specially 100pmol:1mg through biotin labeled described capture probe and the proportioning through the described magnetic bead of marked by streptavidin;
Further, hatch described in be specially 20-30 DEG C (as 25 DEG C) 80-150rpm (as 100rpm) concussion hatch 10-30min (as 15min).Described liquid environment of hatching is binding buffer liquid; The solvent of described binding buffer liquid is water, solute and concentration as follows: Tris 0.605g/L, EDTA 0.185g/L, NaCl 58.45g/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 get respectively the described magnetic bead solution through marked by streptavidin (solvent is described binding buffer liquid) that the solution through biotin labeled described capture probe (solvent is described binding buffer liquid) that concentration is 500nM (with DNA metering) and concentration are 10mg/ml (measuring with magnetic bead), mix according to the proportioning of volume ratio 2:1, 10-30min (as 15min) is hatched in 20-30 DEG C (as 25 DEG C) 80 ~ 150rpm (as 100rpm) concussion, namely described magnetic capture probe is obtained.
In described kit, also can containing the readable carrier (as instructions) recording following (A) and/or (B):
(A) to detect or whether auxiliary detection cell to be measured is the method for 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:10
5individual: 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, after described hatching, also can comprise the step adopting cleaning buffer solution to clean product; The solvent of described cleaning buffer solution is water, solute and concentration as follows: NaCl 8g/L, KCl 0.2g/L, CaCl
20.14g/L, Na
2hPO
4h
2o 0.1g/L, MgCl
26H
2o 1.42g/L, NaHCO
30.35g/L, KH
2pO
40.2g/L, glucose 4.5g/L.
(a2) system after hatching step (a1) carries out magnetic enrichment, and remove supernatant, in enriched product, add TMB nitrite ion and hydrogen peroxide, 37 DEG C of lucifuges hatch 30min;
(a3) determine whether cell to be measured is leukaemia as follows: if step (a2) hatch after system comparatively hatch before compare and there occurs chromogenic reaction (namely reaction system becomes blue by colourless), then described cell to be measured is or candidate is leukaemia; Otherwise, then described cell to be measured be not or candidate for leukaemia;
(B) detect the method for leukaemia's content in cell sample to be measured, comprise the steps:
(B1) acquisition of typical curve:
(b1) suspensions different to described aptamers functionalization Hemin-graphene composite nano material, series standard leukaemia 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, the suspension that series standard leukaemia content is different and described magnetic capture probe are specially according to the proportioning mixing of 25 μ g:20 μ l:25 μ g: the suspension (liquid is described cleaning buffer solution) by described aptamers functionalization Hemin-graphene composite nano material content being the described aptamers functionalization Hemin-graphene composite nano material of 0.5mg/ml, the suspension (liquid is described cleaning buffer solution) that described series standard leukaemia content is different, mix according to the ratio that volume ratio is 25:20:2.5 with the solution (solvent is described cleaning buffer solution) that concentration is the described magnetic capture probe of 10mg/ml (with magnetic bead gauge).In the suspension that series standard leukaemia content described in every 20 μ l is different, the content of described standard leukemia cell is followed successively by 10
5individual, 10
4individual, 10
3individual, 10
2individual, 10
1individual, 0.
As required, after described hatching, also can comprise the step adopting cleaning buffer solution to clean product; The solvent of described cleaning buffer solution is water, solute and concentration as follows: NaCl 8g/L, KCl 0.2g/L, CaCl
20.14g/L, Na
2hPO
4h
2o 0.1g/L, MgCl
26H
2o 1.42g/L, NaHCO
30.35g/L, KH
2pO
40.2g/L, glucose 4.5g/L.
(b2) the some systems after hatching step (b1) carry out magnetic enrichment, and remove supernatant, in enriched product, add TMB nitrite ion and hydrogen peroxide, 37 DEG C of lucifuges hatch 30min;
(b3) the some systems after hatching step (b2) carry out ultraviolet absorption value detection, and determined wavelength is 550-800nm (as 650nm);
(b4) ultraviolet absorption value of the described some systems obtained according to step (b3), and the logarithm of the standard leukemia cell content of correspondence, obtain the typical curve equation between the logarithm of standard leukemia cell content and ultraviolet absorption value;
Wherein, described standard leukemia cell is specially cem cell (human T lymphocyte leukaemia).
(B2) adopt the typical curve equation of step (B1) gained according to comprising the steps that the method for (b5)-(b6) measures the content of leukaemia in cell sample to be measured:
(b5) operate according to step (b1)-(b3), (in cell sample suspension to be measured described in 20 μ l, total cellular score is as being 10 only suspensions different for described series standard leukaemia content to be replaced with cell sample suspension to be measured
5individual), all the other operations are all identical;
(b6) in the typical curve equation that ultraviolet absorption value substitution step (B1) step (b5) recorded obtains, thus the content of leukaemia in described cell sample to be measured is obtained.
In step (a2) and (b2), describedly in enriched product, add TMB nitrite ion and hydrogen peroxide is specially: in enriched product, add TMB nitrite ion described in 100 μ l (as formula is: solvent is DMSO, TMB concentration is 0.6mg/ml) and 1 μ l 500mM hydrogen peroxide.
Described Hemin-graphene composite nano material also belongs to protection scope of the present invention.
The application of described Hemin-graphene composite nano material in the described kit detected for leukaemia of preparation also belongs to protection scope of the present invention.
Described leukaemia can be cem cell.
Kit provided by the present invention is based on Graphene/leukemic bio-sensing method of class peroxidase 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 raises sensitivity and the specificity of the method; (2) using aptamer as identification probe, more stable and economical compared to the antibody in classic method; (3) detection signal naked eyes are visible, without the need to instrument and the complicated operation of costliness; (4) detection time is short, just can complete in one hour, more economical and convenient compared to classic method.To sum up, the target leukaemia of low concentration can catch and enrichment by bio-sensing method of the present invention at short notice, and amplification detection signal, realizes carrying out quick, accurate, sensitive detection to leukaemia.
Accompanying drawing explanation
Fig. 1 is the characteristic absorption peak of Graphene and electrochemical Characterization figure modified through Hemin.A is characteristic absorption peak testing result; B is electrochemical Characterization figure.
Fig. 2 is the principle simulation figure of Graphene of the present invention/leukemic bio-sensing method of class peroxidase dual signal amplification detection.
Fig. 3 is the typical curve that the bio-sensing method of Graphene/class peroxidase dual signal amplification detection leukaemia detects different number cem cell.
Embodiment
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 can obtain from commercial channels.
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.
X-factor (Hemin): sigma Products, its catalog number is 51280 (please indicate).
Embodiment 1, the preparation of kit detected for leukaemia
For containing magnetic capture probe and aptamers functionalization Hemin-graphene composite nano material in the kit that leukaemia detects.
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, is the raw work Products of Chinese Shanghai, and this probe single-stranded DNA sequence used is as follows:
5 '-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGAAAAAA-3 ' (sequence 1), wherein biotin labeling molecule is at 3 ' end of nucleic acid chains.
(2) through the magnetic bead of marked by streptavidin
The magnetic bead of described marked by streptavidin is the Dynabeads Streptavidin MagneSphere M280 that Invitrogen company produces, and the design parameter of this magnetic bead is as follows: as magnetic bead mean diameter 2.8 μm, surface area: 4-8m2/g, density: 1.4g/cm3 (I does not find), magnetic bead content: 10mg/ml, isoelectric point: pH 5.0, low electric charge :-10mV (pH7) (I does not find), CV value <3%, iron content (ferrite): 12% (17%).
(3) preparation of magnetic capture probe
1, be mixed with through biotin labeled capture probe binding buffer liquid the solution that concentration is 500nM (with DNA metering) by what obtain in step ().Wherein, the solvent of described binding buffer liquid is water, solute and concentration as follows: Tris 0.605g/L, EDTA 0.185g/L, NaCl 58.45g/L, HCl regulate pH to 7.5.
2, (concentration is 10mg/ml to get the magnetic bead through marked by streptavidin that 200 μ l steps (two) obtain, calculate with the gauge of magnetic bead), after binding buffer liquid (filling a prescription the same) washing 2 times, then magnetic bead is suspended in the binding buffer liquid of 200 μ l.
3, getting concentration that 400 μ l steps 1 obtain is that (concentration is for 10mg/ml for the magnetic bead of the marked by streptavidin processed through step 2 through biotin labeled capture probe solution and 200 μ l of 500nM (with DNA metering), 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, abandon supernatant, gained precipitation binding buffer liquid (filling a prescription the same) is resuspended.
4, with the binding buffer liquid (filling a prescription the same) of 600 μ l cleaning twice, finally use the resuspended magnetic bead being fixed with DNA of 200 μ l binding buffer liquid (filling a prescription the same) to 10mg/ml (in magnetic bead).
Two, the preparation of Hemin-graphene composite nano material
1, through preparation and the qualification of the Graphene of Hemin modification
(1) through the preparation of the Graphene of Hemin modification
After the X-factor (Hemin) (solvent is DMSO) be graphene oxide suspension (liquid is water) and the 1mL concentration of 1mg/mL by 1mL concentration being 1mg/mL mixes, in mixed system, adding ammoniacal liquor and hydrazine hydrate, (ammoniacal liquor (massfraction 25-28%) added is 10 μ L, hydrazine hydrate (massfraction 80%) is 3 μ L), in 60 DEG C of water-baths 4 hours, vacuum freezedrying obtained the Graphene (being designated as HGNs) modified through Hemin.
(2) through the qualification of the Graphene of Hemin modification
On the one hand, carry out uv absorption blob detection to the Graphene modified through Hemin, concrete operations are as follows: add in cuvette by the Graphene suspension that 100 μ LHemin modify, detect it with ultraviolet spectrophotometer, wavelength coverage is 200-600nm.Experiment arranges graphene oxide, X-factor (Hemin) in contrast simultaneously.
On the other hand, carry out electrochemical signals detection to the Graphene modified through Hemin, concrete operations are as follows: Graphene (concentration is 0.5mg/ml) the 20 μ L modified by Hemin drips on glass-carbon electrode, hatches 12h for 25 DEG C.PBS damping fluid (solvent is water, solute and concentration as follows: NaCl 8g/L, KCl 2g/L, Na
2hPO
4h
2o 3.5g/L, KH
2pO
40.2g/L) middle differential pulse voltametry (DPV) signal to Hemin detects (using saturated calomel electrode as contrast electrode), and potential range is: 0 ~-0.6V.Experiment arranges the glass-carbon electrode of naked glass-carbon electrode and modification graphene oxide in contrast simultaneously.
Characteristic absorption peak testing result is as shown in A in Fig. 1, the characteristic absorption peak of graphene oxide is at about 233nm, the characteristic absorption peak of Hemin is at about 384nm, by reduction, Hemin is loaded to after on Graphene, this absorption peak through the Graphene (HGNs) that Hemin modifies appears at 264nm and 413nm, all there is red shift in the absorption peak compared to graphene oxide and Hemin, illustrates that Hemin successfully loads to graphenic surface.
Electrochemical signals testing result is as shown in B in Fig. 1, all there is not obvious electrochemical signals in the glass-carbon electrode of naked glass-carbon electrode and modification graphene oxide, and the glass-carbon electrode having modified the Graphene (HGNs) modified through Hemin demonstrates the electrochemical signals of Hemin, this galvanochemistry 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 '-GAG AGA GAG AGA GAG AGA GAG AGA GAG AGA ATC TAA CTG CTGCGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3 ' (sequence 2)
Graphene suspension (liquid is water) and the 100 μ L concentration of modifying through Hemin that the step 1 being 1mg/mL by 100 μ L concentration prepares are that (liquid is binding buffer liquid for the aptamers probe solution of 8 μMs, (for 1L solution) composed as follows: Tris 0.605g/L, EDTA 0.185g/L, NaCl 58.45g/L, HCl regulates pH to 7.5) mix after, room temperature (25 DEG C) lucifuge hatches 24 hours, period successively adds aging (each 0.5 μ L NaCl aqueous solution (concentration is 1M) of aqueous solution (final concentration is 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 kit of leukaemia's detection
Kit provided by the present invention realizes detecting based on the leukemic bio-sensing method of Graphene/class peroxidase dual signal amplification detection, and its principle simulation figure as shown in Figure 2.Specifically comprise following content:
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, Hemin-graphene composite nano material and the magnetic capture probe of itself and aptamers probe functionalization form sandwich structure, under the influence of a magnetic field, this sandwich structure is attracted near magnet, now contain a large amount of Graphene/class peroxidase composite nano materials in this sandwich structure, add substrate TMB, chromogenic reaction can occur, utilize ultraviolet-visible pectrophotometer to detect it, obvious uv absorption signal can be obtained.When target cell does not exist, be adsorbed near magnet is magnetic capture probe, a large amount of Graphene/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-visible pectrophotometer, faint uv absorption signal or no signal only can be obtained.Whether there is chromogenic reaction by adding substrate TMB observe system, the qualitative detection to leukaemia can be realized; Utilize the change of uv absorption signal, the quantitative test to leukaemia can be realized; By replacing different capture probes, the method can also realize the detection to other biological mark, cell, metallic ion etc., make it to become a kind of general bioanalytical sensing platform, for the detection of leukaemia provides new approaches, follow the tracks of for the major disease early warnings such as tumour, early diagnosis and curative effect and economic, convenient, practical new technology, new method is provided.
1, the qualitative checking method of kit of the present invention
Whether be leukaemia, specifically comprise the steps: if adopting this kit to detect cell to be measured
(1) get 50 μ l concentration be the obtained aptamers functionalization Hemin-graphene composite nano material suspension of the step 2 of 0.5mg/ml (liquid is cleaning buffer solution, and solvent is water, solute and concentration as follows: NaCl 8g/L, KCl0.2g/L, CaCl
20.14g/L, Na
2hPO
4h
2o 0.1g/L, MgCl
26H
2o 1.42g/L, NaHCO
30.35g/L, KH
2pO
40.2g/L, glucose 4.5g/L), 20 μ l cell (10 to be measured
5individual cell, liquid is cleaning buffer solution) and 2.5 μ l concentration be that the obtained magnetic capture probe suspension (liquid is cleaning buffer solution) of 10mg/ml (with magnetic bead gauge) step one mixes, 37 DEG C of lucifuges hatch 30min;
(2) product is washed twice with cleaning buffer solution (filling a prescription the same), magnetic enrichment is carried out after washing, remove supernatant, 100 μ l TMB nitrite ions (formula: solvent is DMSO is added in enriched product, TMB concentration is 0.6mg/ml) and 1 μ l 500mM hydrogen peroxide mix, 37 DEG C of lucifuges hatch 30min;
(3) determine whether cell to be measured is leukaemia as follows: if step (2) hatch after system comparatively hatch before compare and there occurs chromogenic reaction (namely reaction system becomes 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 kit of the present invention
Adopt this kit to detect leukaemia's content in cell sample to be measured, specifically comprise the steps:
A. the acquisition of typical curve
(1) get Hemin-graphene composite nano material suspension (liquid is described cleaning buffer solution) that 50 μ l concentration are the aptamers functionalization that the step 2 of 0.5mg/ml obtains, 20 μ l cem cell samples (arrange number of cells for as Gradient: 10
5individual, 10
4individual, 10
3individual, 10
2individual, 10
1individual, 0, liquid is described cleaning buffer solution) and 2.5 μ l concentration be that the obtained magnetic capture probe suspension (liquid is described cleaning buffer solution) of 10mg/ml (with magnetic bead gauge) step one mixes, 37 DEG C of lucifuges hatch 30min;
(2) product is washed twice with cleaning buffer solution (filling a prescription the same), magnetic enrichment is carried out after washing, remove supernatant, 100 μ l TMB nitrite ions (formula: solvent is DMSO is added in enriched product, TMB concentration is 0.6mg/ml) and 1 μ l500mM hydrogen peroxide mix, 37 DEG C of lucifuges hatch 30min;
(3) carry out ultraviolet absorption value detection with ultra-violet and visible spectrophotometer to each sample, determined 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 typical curve equation between the logarithm (lg) of cem cell quantity and ultraviolet absorption value (OD650), horizontal ordinate (x) represents the logarithm (lg) of cem cell quantity, and ordinate (y) represents ultraviolet absorption value (OD650);
B. the typical curve equation of steps A gained is adopted to measure the content of leukaemia in cell sample to be measured according to the method comprised the steps:
(5) operate according to (1) in steps A-(3), (total cellular score is 10 only 20 μ l cem cell samples to be replaced with 20 μ l cell sample to be measured
5individual);
(6) OD650 recorded is substituted into the typical curve equation of steps A gained, thus calculate the logarithm of leukaemia's quantity in 20 μ l cell sample to be measured, and then obtain the content of leukaemia in cell sample to be measured.
Embodiment 2, the application example of kit detected for leukaemia
Whether one, adopt the kit of embodiment 1 to detect cell to be measured is leukaemia
Cell sample to be measured: cem cell (people's Pancytopenia cell), Ramos cell (human B lymphocyte oncocyte).
Detect 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, and cem cell group has obvious chromogenic reaction (namely reaction system becomes blue by colourless), and other groups are then the same with blank group, do not have chromogenic reaction to produce.This result shows, kit provided by the present invention can realize the specific detection to leukaemia.
Two, the kit of embodiment 1 is adopted to detect the content of leukaemia in cell sample to be measured
Cell sample to be measured: containing 2 × 10
3suspension (liquid the is described cleaning buffer solution) sample of individual cem cell.
Detect 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 typical curve equation between the logarithm (lg) of cem cell quantity and ultraviolet absorption value (OD650) is y=0.016x+0.0419 (R
2=0.9759), specifically as shown in Figure 3.As seen from the figure, ultraviolet absorption value (OD650) increases with analysis thing cem cell number and increases, 10
1~ 10
5linearly, Monitoring lower-cut can reach 10 cells for the logarithm value of individual cell context inner cell number and light absorption value.
The OD650 of 2, measured cell sample to be measured is 0.095, substitute into typical curve equation, the logarithm (lg) obtaining cem cell quantity is 3.3187, and then to calculate cem cell quantity quantity in cell sample to be measured be 2083, conforms to actual.Visible, kit provided by the present invention can realize the quantitative detection to leukaemia, and testing result accurately and reliably.
Claims (10)
1. for the kit that leukaemia detects, 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 be fixed on described magnetic bead; Described capture probe is the single strand dna shown in sequence in sequence table 1;
(b) aptamers functionalization Hemin-graphene composite nano material;
Described aptamers functionalization Hemin-graphene composite nano material by the Graphene modified through X-factor and be fixed on described through X-factor modify Graphene on aptamers probe form; Described aptamers probe is the single strand dna shown in sequence in sequence table 2.
2. kit according to claim 1, it is characterized in that: described aptamers functionalization Hemin-graphene composite nano material prepares according to the method comprised the steps: be the ratio of 0.5mg:4-40nmol according to the described Graphene modified through X-factor and the proportioning of described aptamers probe, the suspension of the described Graphene through X-factor modification is mixed with the solution of described aptamers probe, 20-25 DEG C of lucifuge hatches 16-24h, obtains described Hemin-graphene composite nano material.
3. kit according to claim 2, it is characterized in that: carrying out during a described 20-25 DEG C lucifuge hatches 16-24h, also comprise and in incubation system, to add NaCl obtain containing NaCl system, NaCl described be the step of 10mM containing the concentration in NaCl system.
4. according to described kit arbitrary in claim 1-3, it is characterized in that: the described Graphene modified through X-factor is 1:(1 ~ 10 by graphene oxide and X-factor according to mass ratio) ratio prepare.
5. kit according to claim 4, it is characterized in that: the described Graphene modified through X-factor prepares according to the method comprised the steps: be 1:(1 ~ 10 by graphene oxide and X-factor according to mass ratio) ratio mixing after, add ammoniacal liquor and hydrazine hydrate, hatch 2 ~ 6h for 60 DEG C, obtain the described Graphene modified through X-factor.
6. according to described kit arbitrary in claim 1-5, it is characterized in that: described magnetic capture probe prepares according to the method comprised the steps: jointly hatch through biotin labeled described capture probe and the described magnetic bead through marked by streptavidin, obtain described magnetic capture probe.
7. kit according to claim 6, is characterized in that: when hatching described in carrying out, and is 100pmol:1mg through biotin labeled described capture probe and the proportioning through the described magnetic bead of marked by streptavidin; Or
Describedly hatch as 10-30min is hatched in 20-30 DEG C of concussion; Or
Described hatching is carried out in binding buffer liquid; The solvent of described binding buffer liquid is water, solute and concentration as follows: Tris 0.605g/L, EDTA 0.185g/L, NaCl 58.45g/L; The pH value of described binding buffer liquid is 7.5.
8. according to described kit arbitrary in claim 1-7, it is characterized in that: also containing the readable carrier recording following (A) and/or (B) in described kit:
(A) to detect or whether auxiliary detection cell to be measured is the method for 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:10
5individual: 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 hatching step (a1) carries out magnetic enrichment, obtains enriched product, in described enriched product, add TMB and hydrogen peroxide, 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 comparatively 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 be not or candidate for leukaemia;
(B) detect the method for leukaemia's content in cell sample to be measured, comprise the steps:
(B1) acquisition of typical curve:
(b1) suspensions different to described aptamers functionalization Hemin-graphene composite nano material, series standard leukaemia 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 hatching step (b1) 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 hatching step (b2) carry out ultraviolet absorption value detection, and determined 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, obtain the typical curve equation between the logarithm of described standard leukemia cell content and described ultraviolet absorption value;
(B2) adopt the typical curve equation of step (B1) gained according to comprising the steps that the method for (b5)-(b6) measures the content of leukaemia in cell sample to be measured:
(b5) operate according to step (b1)-(b3), only suspensions different for described series standard leukaemia content is replaced with cell sample suspension to be measured, all the other operations are all identical;
(b6) in the typical curve equation that ultraviolet absorption value substitution step (B1) step (b5) recorded obtains, thus the content of leukaemia in described cell sample to be measured is obtained.
9. claim 1-5 arbitrary described in aptamers functionalization Hemin-graphene composite nano material.
10. aptamers functionalization Hemin-graphene composite nano material according to claim 9 is preparing the application in claim 1-8 in arbitrary described kit detected for leukaemia.
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