CN101928767A - Electrochemical DNA biosensor for detecting BCR/ABL fusion gene of chronic myeloid leukemia (CML) - Google Patents
Electrochemical DNA biosensor for detecting BCR/ABL fusion gene of chronic myeloid leukemia (CML) Download PDFInfo
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Abstract
The invention provides an electrochemical DNA biosensor for detecting the BCR/ABL fusion gene of chronic myeloid leukemia (CML), which comprises the following steps of: (1) designing and synthesizing the specificity sequence of the CML according to the fusion locus of the universal primer sequence of the selected CML gene type to be detected; and (2) building the electrochemical DNA biosensor by combining a chemical bonding technology, a molecular hybridization technology, a locked nucleic acid probe technology with an electrochemical enzyme linked immunity technology for detecting the BCR/ABL fusion gene of the CML. The biosensor greatly strengthens the sensitivity and the specificity, thereby being capable of realizing the early diagnosis of the chronic myeloid leukemia (CML).
Description
Technical field
The present invention relates to be used to detect the electrochemical DNA biosensor of chronic myelocytic leukemia BCR/ABL fusion gene
Background technology
Chronic myelocytic leukemia, be called for short slow grain (Chronic Myelocytic Leukemia, CML), be with the malignant change of the multipotential stem cell level of acquired chromosome abnormalty and a kind of cell strain disease that causes, its Clinical symptoms is that significant granulocyte excessively generates.Chronic myelocytic leukemia is one of young and middle-aged healthy malignant hematologic disease of serious harm, is type more common in the leukemia.Because leukemia patient tangible symptom do not occur at the initial stage of falling ill, this has brought difficulty to a certain degree for leukemic early diagnosis and detection, and the early diagnosis level will directly influence leukaemic's result of treatment and life quality, and its significance is self-evident.
The methods for clinical diagnosis of CML mainly comprises chromosome analysis, Southern-Blot, FISH, RT-PCR technology etc. at present, and all there is certain limitation in these methods, waste time and energy as chromosome analysis, and sensitivity are low; Though traditional Southern Blot detects high specificity, much less responsive to the detection of low copy gene order, method complexity not only, sense cycle is long, and costs an arm and a leg and have radiocontamination concurrently, has limited widespread use clinically; The RT-PCR complex operation, cost an arm and a leg.Problems such as, complex operation not high as specificity, susceptibility, testing cost height have limited the application in clinical.Therefore, a kind of easy, quick, accurate, sensitive, economic CML gene diagnosis technology of development research has extremely wide application prospect.
Electrochemical DNA biosensor is to adopt electrochemical transducer, detects the biosensor of specific dna sequence (gene) with the electrochemical means selectivity.The review article of relevant electrochemical DNA biosensor has more report, is primarily aimed at electrode (glass-carbon electrode, gold electrode, the carbon paste electrode etc.) immobilization of surperficial ssDNA probe and the screening of highly sensitive electrochemistry hybridization indicator (being marker) and studies.Under suitable temperature, pH, ionic strength, the dna probe molecule of electrode surface can optionally be hybridized with target sequence, forms double-stranded DNA (dsDNA), thereby causes the change of electrode surface structure.Textural difference before and after this hybridization can be discerned by having electroactive hybridization indicator, thereby reaches the purpose that detects target sequence (or specific gene).
Dna probe and complementary interchain interact has influenced the performance of electrochemical sensor to a great extent, and has the problem of poor stability in order to the ssDNA majority of design sensor.So, the dna probe of development highly sensitive and high specific, be applied to and complementary sequence between the detection of hybridization information significant.
Modern molecular biology studies show that slow grain leukemia Ph karyomit(e) is t (9; 22) mutually due to the transposition, its metathesis product BCR/ABL fusion gene betides in the chronic myelocytic leukemia more than 95%, is the marker gene of malignant clone.
Below described for the inventor take the lead in will design chronic myelocytic leukemia BCR/ABL fusion gene probe (5 ' modifies amino or sulfydryl probe, 5 ' modifies amino or sulfydryl hair clip probe, 5 ' modifies amino or sulfydryl lock nucleic acid probe, 3 ' the modification amino or the capture probe of sulfydryl and the signal probe of 5 ' modified biological element), according to different kind of material electrode (gold, glass carbon, carbon paste electrode etc.), select different assembling modes with nucleic acid probe modify electrode surface ((the surface electrode that contains carboxyl with EDC and NHS as the coupling activator with amido modified probe to electrode surface, the gold surface electrode arrives electrode surface with golden sulfide linkage with the sulfydryl modification probe stationary), form ssDNA probe modification electrode; Utilize electrochemistry hybridization indicator (curcumine, rhabarberone, TANSHINONES and acridone derivatives) or electrochemical enzymatic linked immunoassay method again, information change before and after the DNA hybridization is converted into the electrochemical response signal (electric current, voltage or electricity are led/resistance) that can measure changes, thereby realize the pulsating detection of target chronic myelocytic leukemia BCR/ABL fusion gene.
Summary of the invention
Embodiment of the invention technical problem to be solved is to be provided for detecting the electrochemical DNA transmitter of chronic myelocytic leukemia BCR/ABL fusion gene, is intended to solve that existing detection method sensitivity is low, sense cycle is grown, cost an arm and a leg etc.
The embodiment of the invention is achieved in that the electrochemical DNA transmitter that is used to detect chronic myelocytic leukemia BCR/ABL fusion gene, and BCR/ABL fusion gene to be detected comprises the steps:
(1) from the NCBI gene database, searches the position of fusion of BCR/ABL fusion gene, select of the probe use of the base of front and back, site as synthetic, select mutation rate is the highest in the BCL-ABL gene gene fragment as probe, through homology comparison, corresponding sequence is synthesized (5 '-NH as the specific sequence of CML
2(or SH) AGA GTT CAAAAG CCCTTC-3 ', hair clip formula 5 '-NH
2LNA capture probe 5 '-CLGG CCA GTA GCLATCT GAC TTTL G-TTT TTT TTT T-SH-3 ' of (or SH)-(CH2) 6-TTCCAAC TCT CAA TGG CTG CCT CCCGTTGG-3 ', lock nucleic acid 5 '-NH2 (or SH), 3 ' end marking sulfhydryl; Biotin labeled LNA the signal probe 5 '-Biotin-GC ALGA GLTT CLAAALAG CLCC TLTC ALG-3 ' of 5 ' end (L is lock nucleic acid monomer position), wherein to the design of LNA, consider and use minimum lock nucleic acid monomer, but can keep the probe chain rigidity preferably and improve its specificity.
(3) for amino or sulfydryl modification probe, utilize chemical bonding with probe stationary to electrode surface (the surface electrode that contains carboxyl with EDC and NHS as the coupling activator with amido modified probe to electrode surface, the gold surface electrode arrives electrode surface with golden sulfide linkage with the sulfydryl modification probe stationary), form the ssDNA modified electrode.Utilize screening and synthetic electrochemistry hybridization indicator to discern the variation of hybridization front and back electrochemical signals again, optionally detecting target sequence is chronic myelocytic leukemia BCR/ABL fusion gene segment.When the concentration of complementary sequence DNA changed, hybridization information was converted into the indicator electrochemical response signal (electric current, voltage or electricity are led/resistance) that can measure and changes.The concentration of the response signal of indicator and dna segment to be measured is linear within the specific limits, thereby realizes the pulsating detection of target chronic myelocytic leukemia BCR/ABL fusion gene.
(4) for the modification probe that utilizes enzyme-linked immunoassay method, adopt chemical bonding or self-assembly embrane method immobilized capture probes, but encapsulant (sulfydryl hexanol, Thiovanic acid are passed through in the unconjugated site of gold copper-base alloy electrode surface, bovine serum albumin, casein etc.) seal; Under given conditions, capture probe and the 5 ' end that the is fixed on electrode surface signal probe hybridization that is marked with vitamin H can be hybridized with the target complementary DNA; The enzyme (horseradish peroxidase etc.) that will be modified with avidin again utilizes and to be attached to the signal probe that 5 ' end is marked with vitamin H and to get on, utilize the signal amplification of enzyme, by detecting substrate for enzymatic activity (3,3 ', 5,5 '-tetramethyl benzidine etc.) hybridize the difference of front and back electrical signal, thus realize the pulsating detection of target chronic myelocytic leukemia BCR/ABL fusion gene
Compare with existing clinical chronic myelocytic leukemia BCR/ABL fusion gene technology, the sense cycle of this technology is relatively short, highly sensitive, for rapid sensitive diagnosis clinically provides possibility.
Description of drawings
Fig. 1 has made up the mechanism figure of electrochemical DNA biosensor that is used to detect chronic myelocytic leukemia BCR/ABL fusion gene of amino or sulfydryl modification probe for the present invention
Fig. 2 has made up the mechanism figure of electrochemical DNA biosensor that is used to detect chronic myelocytic leukemia BCR/ABL fusion gene of enzyme-linked immunoassay method for the present invention
Fig. 3 is the different target sequential detection figure that the sandwich type electrochemical DNA biosensor of lock nucleic acid modification of the present invention detects the BCR/ABL fusion gene.
Fig. 4 is the electrical signal detection figure that the sandwich type electrochemical DNA biosensor of lock nucleic acid modification of the present invention detects the BCR/ABL fusion gene.
Among Fig. 3: lock nucleic acid probe and complementation fully, single base mismatch or complete mismatched dna hybridization back electric current-time plot: complete complementary DNA (a) in the inspection of sandwich type electrochemical DNA biosensor, single alkali plug mismatched dna (b), complete mismatched dna (c), capture probe (d), background current (e).
Among Fig. 4: (A) the hybridization chain electrochemical signals of different concns relatively; The target dna amount is respectively 10nM from (a) to (h), 5nM, 1nM, 100pM, 10pM, 100fM and 10fM.Inserting picture is the logarithmic plot of electric current and target dna concentration.(B) linear relationship chart (concentration range 10～250fM) of 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 clearer,, the present invention is further elaborated below in conjunction with embodiment.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in qualification the present invention.
The electrochemical DNA transmitter that is used to detect chronic myelocytic leukemia BCR/ABL fusion gene that the embodiment of the invention provides, BCR/ABL fusion gene to be detected comprises the steps:
(1) from the NCBI gene database, searches the position of fusion of BCR/ABL fusion gene, select of the probe use of the base of front and back, site as synthetic, select mutation rate is the highest in the BCL-ABL gene gene fragment as probe, through homology comparison, corresponding sequence is synthesized (5 '-NH as the specific sequence of CML
2(or SH) AGA GTT CAAAAG CCCTTC-3 ', hair clip formula 5 '-NH
2LNA capture probe 5 '-CLGG CCA GTA GCLATCT GAC TTTL G-TTT TTT TTT T-SH-3 ' of (or SH)-(CH2) 6-TTCCAAC TCT CAA TGG CTG CCT CCCGTTGG-3 ', lock nucleic acid 5 '-NH2 (or SH), 3 ' end marking sulfhydryl; Biotin labeled LNA the signal probe 5 '-Biotin-GC ALGA GLTT CLAA ALAG CLCC TLTC ALG-3 ' of 5 ' end (L is lock nucleic acid monomer position).
(2) for amino or sulfydryl modification probe (Fig. 1: 2 probes), utilize chemical bonding with 1 * 10
-6The mol/L probe stationary to electrode surface (Fig. 1: 1 electrode) (the surface electrode that contains carboxyl with 5mmol/L EDC and 8mmol/LNHS be 20 μ l damping fluids of coupling activator with amido modified probe to electrode surface; The gold surface electrode arrives electrode surface with golden sulfide linkage with the sulfydryl modification probe stationary, and unconjugated carat (measure of the purity of gold) point is passed through 1 * 10
-5Mol/L sulfydryl hexanol, 1 * 10
-5The mol/L Thiovanic acid, 0.5% bovine serum albumin, encapsulants such as 0.5% casein seal, and after the sealing, need to adopt detergent washing to remove non-specific adsorption, as 0.2mol/L pH 7.4PBS, 0.2%SDS etc.), form the ssDNA modified electrode.Again under appropriate condition (DNA hybridization solution acidity, hybridization temperature, time, ionic strength), (Fig. 1: 3 electrochemistry hybridization indicators) (curcumine, rhabarberone, TANSHINONES and acridone derivatives) discerned hybridization (Fig. 1: the variation of front and back electrochemical signals 4 target dnas), optionally detecting target sequence is chronic myelocytic leukemia BCR/ABL fusion gene segment to utilize screening and synthetic electrochemistry hybridization indicator.
(3) (SH) lock nucleic acid modification probe (Fig. 2) is an example, adopts the self-assembly embrane method to fix 1 * 10 for the sulfydryl that utilizes enzyme-linked immunoassay method
-6The mol/L capture probe, (capture probe SH) fixes in the characteristic that gold electrode surfaces can form self-assembled monolayer, the unconjugated site of gold electrode surfaces is sealed by encapsulant, as 1 * 10 according to the band sulfydryl
-5Mol/L sulfydryl hexanol, 1 * 10
-5The mol/L Thiovanic acid, 0.5% bovine serum albumin, 0.5% casein etc.After the sealing, need to adopt detergent washing to remove non-specific adsorption, as 0.2mol/L pH 7.4PBS, 0.2%SDS etc.
(4) draw the melting temperature(Tm) curve of LNA-DNA under the same conditions respectively with ultraviolet spectrophotometry.In quartz colorimetric utensil, add certain density LNA probe, then add complementary sequence, room temperature hybridization 30min with concentration.With the temperature rise rate of 0.4 ℃/min, rise to 110 ℃ from 10 ℃, 1 ℃ at every interval records corresponding solution absorbency in the 260nm place, and the line space of going forward side by side is proofreaied and correct in vain, draws absorbancy-temperature curve.The temperature of selecting specific temperature to hybridize as probe and complementary strand;
(5) under specific temperature, be fixed on the capture probe (1 * 10 of electrode surface
-6Mol/L) and 5 ' end is marked with the signal probe (1 * 10 of vitamin H
-6Mol/L) hybridization can realize cohybridization with the target complementary DNA, forms sandwich structure at electrode surface, and contain the end-labelled vitamin H of signal probe in the sandwich structure this moment; Before adding, enzyme also needs unconjugated site is sealed by encapsulant, as the sulfydryl hexanol, and Thiovanic acid, bovine serum albumin, caseins etc. adopt detergent washing to remove non-specific adsorption, as PBS, 0.2%SDS etc. in addition again.
(6) owing to be modified with avidin (avidin) on the horseradish peroxidase (HRP), according to the affinity interaction between avidin and the vitamin H, enzyme (concentration dilution 1: 1000) combination is got on, after the enzyme effect, need to adopt detergent washing to remove non-specific adsorption, as 0.5%Tween 20,0.2mol/L pH 7.4PBS, 0.2%SDS etc.Mainly utilize the signal amplification of enzyme, substrate 3,3 ', 5,5 '-adopt the difference of ampere electric current time curve method research hybridization front and back electrical signal in the tetramethyl benzidine (TMB).
The electrochemical DNA biosensor that the present invention detects chronic myelocytic leukemia BCR/ABL fusion gene has highly sensitive, the characteristics of high specific, and other detection method cost is low relatively.The present invention is adapted to: the research of chronic myelocytic leukemia genotype and cancer-related, the detection that leukemia takes place and develops.
The above only is preferred embodiment of the present invention, and not in order to restriction the present invention, all any modifications of being done within the spirit and principles in the present invention are equal to replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. utilize electrochemical DNA biosensor to detect chronic myelocytic leukemia BCR/ABL fusion gene.
2. be used to detect the electrochemical DNA biosensor of chronic myelocytic leukemia BCR/ABL fusion gene, comprise different kind of material electrode (gold, glass carbon, carbon paste electrode etc.), (5 ' modifies amino or sulfydryl probe to dissimilar nucleic acid probes, 5 ' modifies amino or sulfydryl hair clip probe, 5 ' modifies amino or sulfydryl lock nucleic acid probe, 3 ' the modification amino or the capture probe of sulfydryl and the signal probe of 5 ' modified biological element), dissimilar electrochemistry hybridization indicator (curcumines, rhabarberone, TANSHINONES and acridone derivatives) and the electrochemical enzymatic linked immunoassay method, carry out chronic myelocytic leukemia BCR/ABL fusion gene and measure.It is characterized in that: adopt chemical bonding technology, molecular hybridization, lock Nucleic Acid Probe Technique, the electrochemistry Enzyme-multiplied immune technique making electrochemical DNA biosensor that combines, this transmitter comprises and DNA complementary modification of nucleic acids probe to be measured, by hybridizing with the target complementary DNA, utilize electrochemical analysis method to detect electrochemistry hybridization indicator electrical signal or substrate for enzymatic activity generation electrical signal again, thereby realize mensuration chronic myelocytic leukemia BCR/ABL fusion gene.
3. as being used to detect the electrochemical DNA biosensor of chronic myelocytic leukemia BCR/ABL fusion gene as described in claim 1 and 2, it is characterized in that described modification probe sequence is chronic myelocytic leukemia BCR/ABL fusion gene (5 '-NH
2(or SH) AGA GTT CAAAAG CCC TTC-3 ', hair clip formula 5 '-NH
2(or SH)-(CH2) 6-TTCCAAC TCTCAA TGG CTG CCT CCC GTTGG-3 ', lock nucleic acid 5 '-NH2 (or SH)--A
LGA GTT CA
LA AAG CCC
LLNA the capture probe 5 '-C of TTC-3 ', 3 ' end marking sulfhydryl
LGG CCA GTA GC
LA TCT GAC TTT
LG-TTTTTT TTT T-SH-3 '; Biotin labeled LNA the signal probe 5 '-Biotin-GC A of 5 ' end
LGA G
LTT C
LAAA
LAG C
LCC T
LTC A
LG-3 ' (L is lock nucleic acid monomer position).
4. claim 1,2 and 3 described chemical bondings, molecular hybridization and electrochemical analysis method, comprise the steps: in regular turn to utilize chemical bonding with probe stationary to electrode surface (the surface electrode that contains carboxyl with EDC and NHS as the coupling activator with amido modified probe to electrode surface, the gold surface electrode arrives electrode surface with golden sulfide linkage with the sulfydryl modification probe stationary), form the ssDNA modified electrode.Utilize screening and synthetic electrochemistry hybridization indicator to discern the variation of hybridization front and back electrochemical signals again, optionally detecting target sequence is chronic myelocytic leukemia BCR/ABL fusion gene segment.When the concentration of complementary sequence DNA changed, hybridization information was converted into the indicator electrochemical response signal (electric current, voltage or electricity are led/resistance) that can measure and changes.The concentration of the response signal of indicator and dna segment to be measured is linear within the specific limits, thereby realizes the pulsating detection of target chronic myelocytic leukemia BCR/ABL fusion gene.
5. claim 1,2 and 3 described electrochemical enzymatic linked immunoassay methods, comprise the steps: (1) employing chemical bonding or self-assembly embrane method immobilized capture probes in regular turn, but encapsulant (sulfydryl hexanol is passed through in the unconjugated site of gold copper-base alloy electrode surface, Thiovanic acid, bovine serum albumin, casein etc.) seal; (2) under given conditions, capture probe and the 5 ' end that the is fixed on electrode surface signal probe hybridization that is marked with vitamin H can be hybridized with the target complementary DNA; (3) enzyme (horseradish peroxidase etc.) that will be modified with avidin utilizes and to be attached to the signal probe that 5 ' end is marked with vitamin H and to get on, utilize the signal amplification of enzyme, by detecting substrate for enzymatic activity (3,3 ', 5,5 '-tetramethyl benzidine etc.) hybridize the difference of front and back electrical signal, thus realize the pulsating detection of target chronic myelocytic leukemia BCR/ABL fusion gene.
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