CN105334253A - Method of testing PML/RAR alpha genes through electrochemical biosensor of carbon dot @ graphene oxide composite material - Google Patents
Method of testing PML/RAR alpha genes through electrochemical biosensor of carbon dot @ graphene oxide composite material Download PDFInfo
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Abstract
The invention discloses a method of testing PML/RAR alpha genes through an electrochemical biosensor of a carbon dot @ graphene oxide composite material. The method comprises the steps that (1) a specific probe is designed according to a gene segment to be tested, wherein the PML/RAR alpha fusion genes of acute promyelocytic leukemia are used as an example; (2) capture probes are self-assembled on the C-dots @ GO/GCE surface through amido bonds, and after hybridization of a DNA electrochemical sensor formed with methylene blue (MB) as the electrochemical hybridization indicator with a solution to be tested, whether the solution to be tested contains the PML/RAR alpha fusion genes or not is judged according to difference of electrical signals. The C-dots @ GO nanocomposite prepared through the method has good electrical conductivity, and abundant carboxyl on the GO surface can increase the number of the capture probes; the DNA sensor formed on such basis has the advantages of good selectivity and high sensitivity.
Description
Technical field
The present invention relates to the method for the electrochemical DNA biosensor detection PML/RAR alpha fusion gene based on carbon point graphene oxide composite material, belong to biosensor technique field.
Background technology
Nanometer technology is mainly for a special kind of skill of point subworld be of a size of between 1nm ~ 100nm.The microworld that this size is in atom, molecule is representative and the transitional region that macro object has a common boundary, based on system both atypical microscopic system also atypical macrosystems of this size, therefore unique process based prediction model is had, as surface effect, micro-size effect, quantum effect and macro quanta tunnel effect etc., embody the superior function not available for conventional material.
Biology sensor is made up of molecular recognition part (responsive original paper) and conversion portion (transducer), and going to identify measured target with molecular recognition part, is the main functional component that can cause certain physical change or chemical change.Molecular recognition part is the basis of biology sensor selective determination.The material optionally can offering an explanation predetermined substance in biosome has enzyme, antibody, tissue, cell etc.These molecular recognition function materials can be combined into compound with measured target by identifying, as the combination of antibody and antigen, and the combination of enzyme and matrix.When designing biology sensor, selecting the recognition function material being suitable for determination object, is very important prerequisite.Consider the characteristic of produced compound.Chemical change caused by sensitive element prepared by molecular recognition function material or physical change, go to select transducer, is another important step of development high-quality biology sensor.In sensitive element, the generation or consumption etc. of light, heat, chemical substance can produce corresponding variable quantity.According to these variable quantities, suitable transducer can be selected.
After nanometer technology introduces field of biosensors, improve biology sensor sensitivity and other performance, and inspired Novel Biosensor.Significantly improve because be provided with the transducer of submicron-scale, probe or receive the various performances of micron system biological sensor.
The carbon atom arrangement of Graphene and the monoatomic layer of graphite duplicate, and are that carbon atom is with sp
2hybridized orbital is the individual layer two dimensional crystal that honeycomb lattice arrangement is formed.Because Graphene has very strong toughness, electric conductivity and thermal conductivity, therefore there is good potential using value in a lot of field, can be applicable to a large amount of field such as electronics, optics, biological medicine and daily life future.Although have fine using value in a lot of field, some chemical inertnesses due to itself limit it and further apply.In recent years, a lot of scientific worker is devoted to the Graphene studying functionalization.Graphene oxide is the oxide of Graphene, and by powdered graphite through chemical oxidation and the product after peeling off, after oxidation processes, graphene oxide still keeps the layer structure of Graphene, but introduces many oxygen-containing functional groups on the Graphene monolithic of every one deck.Graphene oxide (GO) is a kind of new carbon of excellent performance, has the functional group that higher specific surface area and surface are abundant.Graphene oxide composite material comprises polymer class compound substance and inorganics class compound substance has a wide range of applications field especially, and therefore the surface modification of graphene oxide becomes another research emphasis.
Carbon point (C-dots) is the spherical carbon nanomaterial of a class size at below 10nm, has the photoluminescence behavior that typical size and excitation wavelength rely on.Have compared with traditional heavy metal quantum dot that size is controlled, good biocompatibility and the advantage such as nontoxic.Carbon point has been widely used in various types of biology sensor as a kind of novel nano material.
Graphene oxide (GO) has larger surface area compared with carbon point, both make a large amount of carbon points be adsorbed on surface of graphene oxide by pi-pi bond effect, the strong electric conductivity of the carboxyl utilizing surface of graphene oxide to enrich and carbon point, has prepared the compound substance of carbon point/graphene oxide.
Acute promyelocytic leukemia (acutepromyelocyticleukemia, APL) is type more common in acute myeloid leukemia (acutemyeloidleukemia, AML), is decided to be acute myelocytic leukemia M by FAB cooperative groups
3type is one of malignant hematologic disease of serious harm human health.The most common chromosome translocation of APL is t (15:17) (q22; Q21), positive rate accounts for 98%, this transposition makes the progranulocyte leukemia (promyelocyticleukemia on No. 15 chromosome, PML) with retinoid receptor II gene (the Retinoicacidreceptor α on No. 17 chromosomes, RAR α) rearrangement, form PML/RAR alpha fusion gene, the PML/RAR alpha fusion protein of its coding has the function of the wild type retinoid receptor being different from normal RAR α allele encodes, can granulocytic differentiation and maturation be blocked, thus cause the generation of APL.Therefore, PML/RAR alpha fusion gene is the specific molecular mark of APL, in the diagnosis of APL and the detection of MRD, have important clinical meaning.
The methods for clinical diagnosis of current APL mainly comprises the hybridization techniques such as chromosome analysis, Southern-Blot, real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH), but these methods all have some limitations: chromosome analysis is wasted time and energy, sensitivity is low; The detection of Southern-Blot to low copy gene order is much less responsive, and not only method is complicated, sense cycle is long, expensive, and has radioactive contamination, limits widespread use clinically; RT-PCR complex operation, expensive; FISH needs to use complicated fluorescence developing system, and relative sensitivity is not high, and clinical large-scale application is difficult to launch.Therefore, research and develop a kind of easy, quick, accurate, sensitive, economic APL gene diagnosis technology and there is application prospect extremely widely.
Described belowly take the lead in preparing graphene oxide/carbon point compound substance for the present inventor, utilize the decorative material that this compound substance is glass-carbon electrode, amido modified specific gene sequences is molecule trapping probe, using methylene blue as electrochemical hybridization indicator, a kind of novel nano electrochemical DNA biosensor built, and for the detection of PML/RAR alpha fusion gene.Carbon point graphene oxide composite material is dripped after being applied to the clean glass-carbon electrode oven dry of process, utilize self assembly embrane method that amido modified capture probe is fixed on C-dotsGO/GCE surface, target gene sequence and capture probe realize hybridizing by base pair complementarity, by the change of strength of current size before and after hybridization, carry out the detection of target gene.Because the glass-carbon electrode of very large specific surface area, strong electric conductivity and biocompatibility C-dotsGO nano-complex modification is as basal electrode, significantly enhance sensitivity and the specificity of detection, thus establish the detection method of highly sensitive, that specificity is good acute promyelocytic leukemia PML/RAR alpha fusion gene, for acute promyelocytic leukemia provides the basis of molecular diagnosis, there is important clinical meaning.
Summary of the invention
The object of the invention is to prepare carbon point graphene oxide novel nanocomposite materials, and with this compound substance modified glassy carbon electrode, propose that a kind of operating process is simple, testing cost is cheap based on this modified electrode and there is high sensitivity and optionally detect the electrochemical DNA biosensor of target gene, and applying the method that this sensor detects PML/RAR α gene.
The object of the present invention is achieved like this, the described method detecting acute promyelocytic leukemia PML/RAR alpha fusion gene based on carbon point graphene oxide composite material assembling electrochemical DNA biosensor, it is characterized in that by preparing carbon point graphene oxide composite material, build electrochemical DNA biosensor, utilize the detection of change realization to target gene of methylene blue strength of current before and after hybridization.
The present invention has prepared the compound substance of carbon point graphene oxide, and described target gene sequence is a fragment of PML/RAR alpha fusion gene, and by the comparison of NCBI gene database homology, this fragment is the distinguished sequence of PML/RAR α.
Describedly to prepare according to the following steps based on carbon point graphene oxide composite material electrochemical DNA biosensor: the preparation of (1) carbon point graphene oxide composite material modified glassy carbon electrode; (2) self assembly of probe forms single stranded DNA, and hybridizes with target gene and form double-stranded DNA; (3) electrochemical DNA hybridization indicator methylene blue enrichment; (4) electrode is in PBS damping fluid, detects target gene by the change of methylene blue strength of current before and after hybridization.
Carbon point graphene oxide composite material modified glassy carbon electrode prepared by the present invention, has large specific surface area, good conduction and DNA load capacity, enhances the sensitivity of this sensor.
Described single, double chain DNA is different from the quantity that methylene blue combines, according to the Difference test target gene of strength of current before and after hybridization.
Getting 8 μ L concentration is that 1 μM of amido modified capture probe drips the C-dotsGO/GCE surface handled well, at room temperature after reaction, dries up, obtain ssDNA/C-dotsGO/GCE electrode with nitrogen after PBS buffer solution for cleaning; The electrode that ssDNA/C-dotsGO modifies is put into 100 μ L to be contained in the hybridization solution of finite concentration target dna, reacts at 35 DEG C, with wash buffer, and the DNA of removing non-specific adsorption, nitrogen dries up, and obtains dsDNA/C-dotsGO/GCE electrode; The C-dotsGO glass-carbon electrode of probe modification is immersed in the MB of 20 μm of ol/LM, stir enrichment 5min, differential pulse voltametry (DPV) curve is recorded after scanning in the PBS solution of blank, DPV scans current potential+0.4V ~-0.6V, amplitude 0.05V, pulse width 0.05s, sampling width 0.0167s, recurrence interval 0.2s, the glass-carbon electrode that time of repose 2s, C-dotsGO modify is 15.9 times, 10.9 times and 20.4 times of naked glass-carbon electrode, the glass-carbon electrode of C-dots modification and the glass-carbon electrode single, double chain current signal difference of GO modification respectively.
A kind of electrochemical DNA biosensor based on the assembling of carbon point graphene oxide composite material of the present invention, comprise glass-carbon electrode, carbon point and graphene oxide composite material, it is characterized in that described being prepared from according to the following steps based on carbon point graphene oxide composite material electrochemical DNA biosensor: (1) by carbon point and graphene oxide composite material in mass ratio 10:1 mix, carbon point graphene oxide composite material is obtained after ultrasonic, (2) carbon point graphene oxide composite material is modified glass-carbon electrode glass-carbon electrode being formed the modification of carbon point graphene oxide compound, (2) glassy carbon electrode surface that capture probe is modified at carbon point graphene oxide compound by amido link self assembly forms single stranded DNA, deposits in case at target dna, capture probe is hybridized by base pair complementarity and target gene and is formed double-stranded DNA, (3) modified electrode before and after hybridization is placed in the enrichment of electrochemical DNA hybridization indicator methylene blue, (4) electrode after enrichment, in PBS damping fluid, detects target gene by the change of methylene blue strength of current before and after hybridization.
Described target gene sequence is a fragment of PML/RAR alpha fusion gene, and this fragment is the distinguished sequence of PML/RAR α.
Described capture probe is 5 '-NH
2-GGTCTCAATGGCTGCCTCCCCG-3 ', target gene is 5 '-CGGGGAGGCAGCCATTGAGACC-3 '.
Specifically, a kind of nano electrochemical DNA biosensor detecting acute promyelocytic leukemia PML/RAR alpha fusion gene of the present invention, testing gene is PML/RAR alpha fusion gene, comprises the steps:
(1) from NCBI gene database, PML/RAR alpha fusion gene sequence is searched, one section of probe as Prof. Du Yucang is wherein selected to use, probe length is 24 bases, through homology comparison, the specific sequence of the sequence of correspondence as PML/RAR α is synthesized, capture probe (5 '-NH
2-GGTCTCAATGGCTGCCTCCCCG-3 '), target sequence (5 '-CGGGGAGGCAGCCATTGAGACC-3 ').
(2) glass-carbon electrode (GCE) is successively 1.0, the Al of 0.3,0.05 μm
2o
3in powder, polishing, polishing, then put into volume ratio V respectively
hNO3: V
h2Ofor the HNO of 1:1
3solution, absolute ethyl alcohol and deionized water for ultrasonic also use N
2dry up.With liquid-transfering gun, carbon point graphene oxide composite material (C-dotsGO) of 8 μ L is dropped in the clean GCE surface of process, put into exsiccator until dry tack free.
(3) capture probe passes through glass-carbon electrode (C-dotsGO/GCE) surface that amido link self assembly is modified at carbon point graphene oxide compound, and deposit in case at target dna, capture probe is combined with target dna by base pair complementarity.
(4) be placed in containing methylene blue (MB) solution enrichment certain hour by the modified electrode before and after hybridization, MB in electrode surface generation redox reaction, thus produces electrochemical signals.In electrochemical detection, galvanochemistry three-electrode system: glass-carbon electrode is working electrode, platinum electrode is to electrode, the saturated KCl of Ag/AgCl() and be contrast electrode, electrochemical measuring technique used is differential pulse voltametry.
The features and advantages of the invention are: compared with prior art, the present invention has prepared carbon point graphene oxide (C-dotsGO) Novel composite nano material modified glassy carbon electrode, the carboxyl that the specific surface area utilizing graphene oxide huge, surface are enriched strengthens the transfer ability of electronics to the strong electric conductivity of the charge capacity and carbon point that improve capture probe, drastically increase the sensitivity of detection.The diagnosis that method of the present invention is expected to be applied to various gene clinically detects, for quick diagnosis provides possibility clinically.And the required detection operating process of this technology is simple, testing cost is cheap, result is sensitive and accurate, is conducive to promoting the use of.
Accompanying drawing explanation
Fig. 1 is the method testing process flow diagram detecting acute progranulocyte PML/RAR alpha fusion gene based on carbon point stannic oxide/graphene nano compound substance electrochemical DNA biosensor of the present invention, in figure: capture probe is labeled as 1, target-probe is labeled as 2, carbon point is labeled as 3, and methylene blue is labeled as 4.
Fig. 2 is carbon of the present invention some phenogram; In figure: (A) transmission electron microscope picture, (B) grain size distribution, (C), (D) high resolution TEM figure, (E) uv absorption spectra, (F) fluorescence spectrum figure.
Fig. 3 is compound substance phenogram of the present invention; In figure: the scanning electron microscope (SEM) photograph of (A) graphene oxide, (B) scanning electron microscope (SEM) photograph of carbon point graphene oxide composite material, (C) transmission electron microscope picture of graphene oxide, (D) transmission electron microscope picture of carbon point graphene oxide composite material, the fluorescence radiation picture (right side) of carbon point (left side), carbon point graphene oxide composite material under (E) uviol lamp (365nm).
Fig. 4 is that different proportion of the present invention prepares compound substance modified electrode figure; In figure: (A) cyclic voltammogram and (C) AC impedance figure: (a) GO; (b) 50:1; (c) 30:1; (d) 10:1; (e) 5:1; (f) 3:1, the peak current figure in (B) corresponding A, AC impedance figure in (D) corresponding C.
Fig. 5 is the differential pulse voltammogram of different materials modified glassy carbon electrode of the present invention; In figure: (A) C-dotsGO/GCE (a), ssDNA/C-dotsGO/GCE (b), dsDNA/C-dotsGO/GCE (c); (B) GO/GCE (d), ssDNA/GO/GCE (e), dsDNA/GO/GCE (f); (C) C-dots/GCE (g), ssDNA/C-dots/GCE (h), dsDNA/C-dots/GCE (i); (D) GCE (j), ssDNA/GCE (k), dsDNA/GCE (l).
Fig. 6 is the AC impedance figure of sensor assembling process of the present invention; In figure: (a) naked GCE, (b) C-dotsGO/GCE, (c) ssDNA/C-dotsGO/GCE, (d) dsDNA/C-dotsGO/GCE.
Fig. 7 is the differential pulse voltammogram after DNA probe of the present invention is hybridized with complete complementary, single base mismatch and complete mismatched dna respectively; In figure: (a) complete complementary DNA, (b) single base mismatch DNA, (c) be mismatched dna completely, and (d) capture probe is not hybridized.Illustration is corresponding current value histogram.
Fig. 8 is the electric signal figure after the object chain of sensor of the present invention and variable concentrations is hybridized; In figure: target dna amount from (a) to (e) is respectively 2.25,1.75,1.25,0.75 and 0.25nmol/L.Wherein insert the linear relationship chart that figure is strength of current and target DNA concentration.
Embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearly understand, below in conjunction with embodiment and accompanying drawing, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The embodiment of the present invention is the (see figure 1) realized like this, and the electrochemical DNA biosensor based on carbon point graphene oxide composite material detects the method for acute promyelocytic leukemia PML/RAR α gene, comprises the steps:
Embodiment 1:
The design procedure of PML/RAR alpha fusion gene probe is as follows:
(1) from NCBI gene database, PML/RAR alpha fusion gene sequence is searched, one section of probe as Prof. Du Yucang is wherein selected to use, probe length is 24 bases, through homology comparison, the specific sequence of the sequence of correspondence as PML/RAR α is synthesized, capture probe (5 '-NH
2-GGTCTCAATGGCTGCCTCCCCG-3 '), target dna sequence (5 '-CGGGGAGGCAGCCATTGAGACC-3 ').
Embodiment 2:
The electrochemica biological sensor preparation process of carbon point graphene oxide composite material is as follows
:
(1) glass-carbon electrode (GCE) is successively 1.0, the Al of 0.3,0.05 μm
2o
3polish in powder, polishing.Then (namely concentration is the HNO of 16mol/L to be respectively put into 1:1 salpeter solution
3be 1:1 with the volume proportion of water), ultrasonic and dry up with nitrogen in absolute ethyl alcohol and distilled water, for subsequent use.
(2) carbon point (being called for short C-dots) characterization result is shown in Fig. 2.As shown in the A in Fig. 2, the carbon point almost spherical prepared, adds up according to the particle diameter of TEM data to carbon point, obtains particle diameter (B see in Fig. 2) within the scope of 0.97 ~ 2.18nm.From the high-resolution-ration transmission electric-lens of carbon point as shown in the D in C and Fig. 2 Fig. 2, the carbon point of preparation has patterned features clearly, and grating constant is 0.203nm, and this characteristic constant is by the sp of graphitic carbon
2hydridization produces.The uv-visible absorption spectra of carbon point is as shown in the E in Fig. 2, and the carbon point of preparation has a more weak ultraviolet absorption peak at 230nm place, and this absorption peak is carbon point conjugated structure π-π transition absorption; From shown in the carbon point fluorescence spectrum figure of the F Fig. 2, prepare carbon point and have and comparatively symmetrical excite (1) and emission spectrum (2), maximum excitation wavelength and emission wavelength are respectively 345nm and 455nm, and Stokes shift is 90nm.
Mixed with certain proportion with graphene oxide (being called for short GO) by carbon point (being called for short C-dots), ultrasonic 30min obtains carbon point graphene oxide composite material.Carbon point graphene oxide composite material characterization result is shown in Fig. 3.As shown in the B in A and Fig. 3 in Fig. 3, graphene oxide has more fold at electrode surface, and after forming compound substance with carbon point, fold obviously reduces; D in C and Fig. 3 in Fig. 3 is transmission electron microscope (TEM) figure of GO and C-dotsGO compound substance respectively.As can be seen from the D figure in Fig. 3, surface of graphene oxide has much spherical carbon point, and roughness also obviously increases compared with simple graphene oxide; And under uviol lamp 365nm light source irradiation, carbon point aqueous solution has stronger blue-fluorescence (be colourless see the left side color in the E in Fig. 3, E figure, the right color is blue); After adding graphene oxide in carbon point aqueous solution, the fluorescence of carbon point disappears.Experimental result shows, carbon point and graphene oxide are combined by electrostatic and pi-pi accumulation interaction and define carbon point stannic oxide/graphene nano compound substance (being called for short C-dotsGO nano composite material).
The C-dotsGO Nanocomposite solution accurately pipetting 8 μ L drips the GCE surface handled well in step (1), puts into exsiccator evaporation drying 30min, obtains the glass-carbon electrode (being called for short C-dotsGO/GCE) that C-dotsGO modifies, for subsequent use.
(3) the iron cyanogen electricity C-dotsGO modified electrode that step (2) prepares being placed in respectively the potassium ferricyanide containing 1mmol/L and 10mmol/L is to solution (this solution is containing 0.1mol/L potassium chloride), and the compound substance modification sensor interface chemical property formed GO and C-dots of different proportion carries out investigation and the results are shown in Figure 4.Shown in the B in A and Fig. 4 Fig. 4, along with C-dots in compound substance measures the increase of ratio, the peak point current of cyclic voltammetry curve from a to f increases gradually, illustrates that carbon point can strengthen the electric conductivity of graphene oxide.And corresponding electrochemical AC impedance test result is as shown in the D in C and Fig. 4 in Fig. 4, increase along with C-dots measures ratio, modified electrode interface impedance value reduces (from curve a to curve f) gradually, and show the increase along with C-dots ratio in compound substance, its electric conductivity strengthens gradually.Above experimental result all shows that carbon point can strengthen the conductive capability of carbon point graphene oxide composite material, and when the mass ratio of ratio GO and C-dots reaches 10:1, enhancing tends towards stability.In order to ensure charge capacity that on compound substance, DNA is larger and stronger electric conductivity, the mass ratio of experimental selection GO and C-dots is that 10:1 prepares C-dotsGO compound substance.
(4) the C-dotsGO modified electrode that step (2) or (3) prepare is placed in the PBS buffer solution containing 20mmol/LEDC and 20mmol/LNHS, after activation 30min, ultrapure water, nitrogen dries up.Then, drip the ssDNA probe of 8 μ L at electrode surface, at room temperature self assembly 2h, ultrapure water, nitrogen dries up.Obtain ssDNA/C-dotsGO/GCE electrode, for subsequent use.
(5) the ssDNA/C-dotsGO modified electrode that step (4) prepares is placed in containing the enrichment of methylene blue (MB) solution.The combination of MB and single stranded DNA is by the combination with the G base specific on single stranded DNA, when hybridization is formed after duplex DNA, because bases G is embedded in double-spiral structure inside, have impact on the affinity of itself and MB to a great extent, so its DPV recovering signal is than little before hybridization after hybridization.By the DPV signal difference before and after hybridization, identifiable design ssDNA and dsDNA, the results are shown in Figure 5.As shown in the D in Fig. 5, curve (k) is the DPV figure of ssDNA modified glassy carbon electrode, and curve (l) is the DPV figure of dsDNA modified glassy carbon electrode, and the current differential of single, double chain DNA on naked glass-carbon electrode is 0.227 μ A; Curve (h) in C figure in Fig. 5 is the DPV figure that ssDNA modifies C-dots glass-carbon electrode, and curve is (i) the DPV figure that dsDNA modifies C-dots glass-carbon electrode, and the current differential of single, double chain DNA on C-dots modified glassy carbon electrode is 0.177 μ A; Curve (e) in B figure in Fig. 5 is the DPV figure that ssDNA modifies GO glass-carbon electrode, and curve (f) is the DPV figure that dsDNA modifies GO glass-carbon electrode, and the current differential of single, double chain DNA on GO modified glassy carbon electrode is 0.25 μ A; Curve (b) in A figure in Fig. 5 is the DPV figure that ssDNA modifies C-dotsGO glass-carbon electrode, and curve (c) is the DPV figure of dsDNA modified glassy carbon electrode, and the current differential of single, double chain DNA on C-dotsGO modified glassy carbon electrode is 3.61 μ A.The glass-carbon electrode that C-dotsGO modifies is 15.9 times, 10.9 times and 20.4 times of naked glass-carbon electrode, the glass-carbon electrode of C-dots modification and the glass-carbon electrode single, double chain current signal difference of GO modification respectively.Show based on the larger hybridization efficiency that improve DNA of the glass-carbon electrode biology sensor of C-dotsGO modification.
(6) the ssDNA/C-dotsGO modified electrode that step (4) prepares is placed in [Fe (CN)
6]
3-/4-in solution, adopt AC impedance electrochemical technology to prepare sensor interface probe assembling process to step (4) and characterize.Characterization result as shown in Figure 6.At [Fe (CN)
6]
3-/4-in solution, curve a is the electrochemical alternate impedance spectrum figure of naked GCE, and its half circular diameter is very little, and resistance value (Ret) is very little; After C-dotsGO modifies GCE, Ret obviously increases (curve b), because C-dotsGO surface is containing abundant-COO
-with [the Fe (CN) in solution
6]
3-/4-producing repulsive interaction makes electron transmission slow down, and causes resistance value to become large; When ssDNA is assembled into C-dotsGO modified electrode surperficial, R
etfurther increase (curve c), mainly because ssDNA has electronegative phosphate backbones, will repel electronegative [Fe (CN)
6]
3-/4-arrive electrode surface and carry out redox reaction, after hybridizing with target dna, electronegative phosphate backbones increases, and hinders the ability of electron transmission to strengthen, R
etbecome large (curve d) again; Show that the different interfaces of this sensor assembling process successfully construct.
(7) the selectivity (see figure 7) of the DNA sensor based on the assembling of C-dotsGO compound substance is investigated.DNA probe respectively with fully-complementary sequence (a), single base mismatch sequence (b), number of base mismatch (c), not containing the hybridization of object chain solution (d) DNA chain, by DPV method to change in electric before and after Probe Hybridization, the specificity of carrying out sensor is investigated.After hybridizing with the target dna of complete complementary, its current signal value is starkly lower than the DNA chain with other mispairing, illustrates that this structure sensor can effective identification form base mispairing recognition capability, has good specificity.
(8) utilize DPV method, the electrochemical DNA biosensor based on the assembling of C-dotsGO compound substance quantitatively detects the complementary ssDNA of acute promyelocytic leukemia PML/RAR α gene target, the results are shown in Figure 8.Between concentration 20pmol/L ~ 200nmol/L, along with the increase of complementary strand concentration, its reduction peak current intensity declines on the contrary.When concentration is greater than 2nmol/L, its peak current intensity remains unchanged substantially, shows that the probe being fixed on electrode surface is hybridized (A in Fig. 8) completely.In 0.25 ~ 2.5nmol/L concentration range, the concentration of strength of current and target dna is good linear relationship (B in Fig. 8), and regression equation is: Δ I (μ A)=7.954-0.5849C
tDNA(nmol/L), R
2=0.986; Detectability reaches 0.083nmol/L (S/N=3).Result shows, the electrochemical DNA biosensor built based on C-dotsGO compound substance has higher sensitivity, and this is that the carboxyl enriched due to C-dotsGO electrode surface effectively improves the probe amount that is fixed on its surface and C-dots makes its conductive capability strengthen thus improves the detection sensitivity of sensor.
The inventive method can be applicable to various gene diagnosis clinically and detects, for quick diagnosis provides possibility clinically.The required detection operating process of this technology is simple, testing cost is cheap and testing result has the advantage such as high sensitivity and selectivity, is conducive to promoting the use of.
The foregoing is only exemplary embodiments of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. one kind is detected the method for acute promyelocytic leukemia PML/RAR alpha fusion gene based on carbon point graphene oxide composite material assembling electrochemical DNA biosensor, it is characterized in that by preparing carbon point graphene oxide composite material, build electrochemical DNA biosensor, utilize the detection of change realization to target gene of methylene blue strength of current before and after hybridization.
2. the method detecting acute promyelocytic leukemia PML/RAR alpha fusion gene based on carbon point graphene oxide composite material electrochemical DNA biosensor according to claim 1, it is characterized in that the compound substance having prepared carbon point graphene oxide, and described target gene sequence is a fragment of PML/RAR alpha fusion gene, by the comparison of NCBI gene database homology, this fragment is the distinguished sequence of PML/RAR α.
3. the method detecting acute promyelocytic leukemia PML/RAR alpha fusion gene based on carbon point graphene oxide composite material electrochemical DNA biosensor according to claim 1 and 2, prepares based on carbon point graphene oxide composite material electrochemical DNA biosensor described in it is characterized in that: the preparation of (1) carbon point graphene oxide composite material modified glassy carbon electrode according to the following steps; (2) self assembly of probe forms single stranded DNA, and hybridizes with target gene and form double-stranded DNA; (3) electrochemical DNA hybridization indicator methylene blue enrichment; (4) electrode is in PBS damping fluid, detects target gene by the change of methylene blue strength of current before and after hybridization.
4. the method detecting acute promyelocytic leukemia PML/RAR alpha fusion gene based on carbon point graphene oxide composite material electrochemical DNA biosensor according to claim 3, it is characterized in that the carbon point graphene oxide composite material modified glassy carbon electrode prepared, there is large specific surface area, good conduction and DNA load capacity, enhance the sensitivity of this sensor.
5. the method detecting acute promyelocytic leukemia PML/RAR alpha fusion gene based on carbon point graphene oxide composite material electrochemical DNA biosensor according to claim 3, it is characterized in that single, double chain DNA is different from the quantity that methylene blue combines, according to the Difference test target gene of strength of current before and after hybridization.
6. the method detecting acute promyelocytic leukemia PML/RAR alpha fusion gene based on carbon point graphene oxide composite material electrochemical DNA biosensor according to claim 3, it is characterized in that getting 8 μ L concentration is that 1 μM of amido modified capture probe drips the C-dotsGO/GCE surface handled well, at room temperature after reaction, dry up with nitrogen after PBS buffer solution for cleaning, obtain ssDNA/C-dotsGO/GCE electrode; The electrode that ssDNA/C-dotsGO modifies is put into 100 μ L to be contained in the hybridization solution of finite concentration target dna, reacts at 35 DEG C, with wash buffer, and the DNA of removing non-specific adsorption, nitrogen dries up, and obtains dsDNA/C-dotsGO/GCE electrode; The C-dotsGO glass-carbon electrode of probe modification is immersed in the MB of 20 μm of ol/LM, stir enrichment 5min, differential pulse voltametry (DPV) curve is recorded after scanning in the PBS solution of blank, DPV scans current potential+0.4V ~-0.6V, amplitude 0.05V, pulse width 0.05s, sampling width 0.0167s, recurrence interval 0.2s, the glass-carbon electrode that time of repose 2s, C-dotsGO modify is 15.9 times, 10.9 times and 20.4 times of naked glass-carbon electrode, the glass-carbon electrode of C-dots modification and the glass-carbon electrode single, double chain current signal difference of GO modification respectively.
7. the electrochemical DNA biosensor based on the assembling of carbon point graphene oxide composite material, comprise glass-carbon electrode, carbon point and graphene oxide composite material, it is characterized in that described being prepared from according to the following steps based on carbon point graphene oxide composite material electrochemical DNA biosensor: (1) by carbon point and graphene oxide composite material in mass ratio 10:1 mix, obtain carbon point graphene oxide composite material after ultrasonic, carbon point graphene oxide composite material is modified glass-carbon electrode glass-carbon electrode being formed the modification of carbon point graphene oxide compound by (2); (2) glassy carbon electrode surface that capture probe is modified at carbon point graphene oxide compound by amido link self assembly forms single stranded DNA, deposits in case at target dna, capture probe is hybridized by base pair complementarity and target gene and is formed double-stranded DNA; (3) modified electrode before and after hybridization is placed in the enrichment of electrochemical DNA hybridization indicator methylene blue; (4) electrode after enrichment, in PBS damping fluid, detects target gene by the change of methylene blue strength of current before and after hybridization.
8. according to claim 7 based on carbon point graphene oxide composite material assembling electrochemical DNA biosensor, it is characterized in that described target gene sequence is a fragment of PML/RAR alpha fusion gene, this fragment is the distinguished sequence of PML/RAR α.
9. according to claim 7 or 8 based on carbon point graphene oxide composite material assembling electrochemical DNA biosensor, it is characterized in that capture probe is 5 '-NH
2-GGTCTCAATGGCTGCCTCCCCG-3 ', target gene is 5 '-CGGGGAGGCAGCCATTGAGACC-3 '.
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