CN107723340A - A kind of electrochemical sensor for CYP2C19*2 detections - Google Patents

A kind of electrochemical sensor for CYP2C19*2 detections Download PDF

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CN107723340A
CN107723340A CN201710803959.2A CN201710803959A CN107723340A CN 107723340 A CN107723340 A CN 107723340A CN 201710803959 A CN201710803959 A CN 201710803959A CN 107723340 A CN107723340 A CN 107723340A
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electrode
cyp2c19
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于超
张成丽
赵璘
赵一璘
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Chongqing Medical University
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Abstract

The present invention relates to clopidogrel associated metabolic gene --- the preparation method and application of the electrochemical sensor of CYP2C19*2 allele detection, belong to technical field of electrochemical detection.It is characterized in that：Synthesis obtains c C first60/CeO2Composite, then nano platinum particle is reduced in c C60/CeO2On composite, then single stranded DNA signal probe is mixed with the composite, bio signal probe is made；Then AuNPs@Fe MOF are passed through, Avidin, LBL self-assembly is used for the fixation of biotinylated DNA capture probes, and so as to be prepared for the electrochemical sensor of CYP2C19*2 allele detection, the sensor is successfully used in the detection that single base mutation occurs for CYP2C19*2 genes.The advantage of the invention is that high sensitivity, high specificity, detection is rapid, convenient.The present invention provides new detection method for clopidogrel associated metabolic gene and instructs individualized treatment.

Description

A kind of electrochemical sensor for CYP2C19*2 detections
Technical field：
The present invention relates to a kind of system of the electrochemical sensor of clinically quantitative Diagnosis CYP2C19*2 allele detection Preparation Method and application, the interlayer type biology sensor that fullerene nano composite material is prepared as signal probe is based especially on, For detecting CYP2C19*2 allele, belong to field of electrochemical detection.
Background technology：
Clopidogrel is one of medicine for treating thrombus thing being most widely used at present, available for treatment atherosclerotic heart and brain Vascular diseases include cerebral arterial thrombosis, myocardial infarction and peripheral arterial disease, are acute coronary syndrome (ACS) and percutaneous hat The conventional medicine of clinical prognosis occurs, improved for the postoperative prevention ischemic events of shape arterial Interventional Therapy (PCI).Clopidogrel conduct A kind of pro-drug, active metabolite need to be changed into through Cytochrome P450 (CYP) and play antiplatelet effects.In clinical practice Aspect, clopidogrel use is very special, if excessive concentration will cause bleeding to its active component in blood, and concentration is too low The side effects such as blood vessel embolism can then be caused.The concentration level of active component and individual inheritance background are close in blood for clopidogrel It is related.Thus, the blood concentration of clopidogrel active metabolite can be reduced by influenceing the gene mutation of CYP functions reduction, so as to The effectiveness of clopidogrel is limited, severe patient produces side effect.Largely research shows, CYP2C19 gene mutation and CYP work( It can reduce relevant.Wherein, the clopidogrel activated product of CYP2C19*2 and low concentration is closely related.Reason mainly has two sides Face, on the one hand, CYP2C19 functions are lost (LOF) genotype [particularly CYP2C19*2, c.681G (rs4244285, ＞ A)] and taken Active metabolite level with clopidogrel in person's body is relatively low.On the other hand, the research of accumulation shows, CYP2C19*2 distribution Frequency is higher than other gene mutations.
At present, clinical application standard is instructed, modern is most accurately detection genotype.In 2011 in U.S.'s food and medicine Management board (Food and Drug Administration, FDA) sends black surround warning to clopidogrel：Clopidogrel metabolism compared with Slow person can not effectively be translated into activated product thus can not give full play to antiplatelet effect, and medical worker should be according to trouble The situation of person carries out CYP2C19 genetic tests, saltant type patient is considered from other antiplatelet drugs or adjustment chlorine pyrrole lattice The therapeutic dose of thunder.Therefore, effective detection CYP2C19*2 genotype is advantageous to instruct the medicine individualized treatment, pushes away simultaneously Enter the development of accurate medical science.At present, it has been reported that detection CYP2C19*2 routine techniques have DNA sequencing, real-time quantitative polymerization Enzyme chain reaction (qPCR) and restriction fragment length polymorphism analysis (PCR-RFLP).However, there is sample treatment in these methods It is cumbersome, cost is high, needs high precision apparatus, it is impossible to realize to the fast quantitative analysis of the gene.Therefore, finding has high sexual valence Realize that to CYP2C19*2 Quantitative detection be current urgent problem than, super-sensitive detection method.
In electrochemica biological sensor analytical technology, in order to improve the sensitivity of detection and shorten the response time, meet The clinically quantitative quick detection of trace materials, at present more using the sensor response patterns of " sandwich " type.Its principle is base In the analysis side of structure load capture probe (CP)-measured target DNA (TP)-marking signal probe (SP) sandwich biological composite Method.Wherein, signal probe label primarily serves enhancing, amplified signal, the effect for improving detection sensitivity, therefore light with foot is lifted The status of weight.Fullerene (the c-C using carboxylated is intended in this experiment60), ceria (CeO2), nano platinum particle (PtNPs) close Cheng Xin signal nano material compound, for immobilized signal probe, electric signal is produced by catalyzing hydrogen peroxide to realize letter Number amplification, finally realize the detection of trace amount DNA in blood.Ceria (CeO2) nano particle is due to its stronger energy of adsorption Power, unique auxiliary catalysis performance and electronics transfer effect between electrode and electrode modified material can be strengthened and by very big Pay close attention on ground.Due to above-mentioned advantage, CeO2It is widely used to catalytic field.However, CeO2Stability in biology sensor is also Have much room for improvement.In order to solve this problem, using c-C60, this stability is high for this experiment, and specific surface area is big, good mechanical property, The nano material of good dispersion.It can be used as the immobilized more CeO of carrier2, and CeO can be improved2Stability.This Outside, in order to give full play to CeO2Auxiliary catalysis effect, this problem introduce PtNPs.Due to its have significant electro catalytic activity, To the strong absorption affinity of amino and thiol group, it can be not only used for catalyzing hydrogen peroxide amplification electrochemical signals, and can be with Immobilized amido modified signal probe.Generally speaking, CeO2There is the effect of concerted catalysis hydrogen peroxide with PtNPs, so as to realize Signal amplification, c-C60In immobilized more CeO2While with PtNPs, the stability of signal material is added.And because There is PtNPs presence, can also be combined with signal probe, be can be used for by the specific binding of signal probe and target sequence Build sandwich type biology sensor.
The project establishes a simple, quick detection method and realized to the special of CYP2C19*2, hypersensitive inspection Survey.The guidance method of personalized medicine is provided for clinical treatment cardiovascular related diseases.
The content of the invention：
1. the purpose of the present invention is the preparation for detecting the electrochemical DNA biosensor of CYP2C19*2 allele Method and application, provide for the medication of clinical clopidogrel and instruct thinking, promote the development of accurate medical science, its feature is including following Step：
(1) fullerene (c-C of carboxylated60)-ceria (CeO2)-nano platinum particle (PtNPs)-ssDNA detections are visited The preparation of pin；
(2) electrochemical DNA biosensor is established, determines CYP2C19*2 genes, draws standard curve.
2. c-C of the present invention60-CeO2The preparation process of-PtNPs-ssDNA compounds specifically includes following steps, its Feature comprises the following steps：
(1)c-C60-CeO2The preparation of nano composite material：
First, by 10mg c-C60It is dissolved in 5mL ultra-pure waters, is placed in and is ultrasonically treated 15min at room temperature, obtains uniform Suspension.Then, by 10mL 0.025M Ce (NO3)3·6H2O mixes with 10mL 0.025M HTMA.Then by two kinds of solution Mix and be maintained in 80 DEG C of water-bath 5 hours, after room temperature cooling with ultra-pure water centrifuge washing for several times.Then, will be prepared Sediment is dried at 60 DEG C, is used for next step.Then, by 20mg c-C60/CeO2Compound is dispersed in 5mL ethanol, is surpassed Sonication 15 minutes, obtains dispersion soln.Then, 0.1mL APTES is mixed with above-mentioned solution, be placed under the conditions of 70 DEG C Kept for 1.5 hours.After being cooled to room temperature, with ultra-pure water centrifuge washing, obtained c-C60/CeO2Complex.Most prepare at last Product is dried at 50 DEG C, is further used.
(2)c-C60-CeO2The preparation of-PtNPs nano composite materials：
Pass through NaBH4Reducing process has synthesized c-C60/CeO2/PtNPs.First, to 1mL c-C60/CeO2Solution (2mg/ ML 1mL H is added in)2PtCl6(1%), and it is ultrasonically treated 5 minutes.Then under agitation by 2mL NaBH4(0.1M) is molten Drop is added in mixture, is reacted 30 minutes, is then centrifuged for washing 3 times, and be dissolved in 1mL ultra-pure water and further use.
(3)c-C60-CeO2The preparation of-PtNPs-ssDNA compounds
By prepared 1mL c-C60/CeO2Signal probe (SP) amido modified with 200 μ L (1 μM)/PtNPs mixes, And it is gently mixed at 4 DEG C 24 hours.Then, after 1mg BSA are mixed with 1mLTE buffer solutions, it is added to c-C60/CeO2/ In PtNPs solution, continue to be gently mixed at 4 DEG C 4 hours, for blocking nonspecific binding site.Then, with 8000rpm Centrifuge c-C60/CeO2/ PtNPs/SP/BSA bioconjugates, washed 3 times with PBS, being dispersed in 1mL hybridization solutions further makes With.
3. according to claim 1 establish electrochemical DNA biosensor, CYP2C19*2 genes are determined, draw mark Directrix curve, it is characterised in that comprise the following steps：
(1) respectively with 0.3 and 0.05 μm of Al2O3Powder by polishing electrode into minute surface, then respectively by ultra-pure water, anhydrous Order each 5min of ultrasound electrode of ethanol, ultra-pure water, drying at room temperature are standby；
(2) 6 μ L, the metal organic frame (AuNPs@Fe-MOFs) of electrode modified material golden nanometer particle@iron are added dropwise Electrode surface, drying at room temperature.
(3) Avidin is incorporated in dried electrode surface (4 DEG C of 12h).
(4) electrode washing is totally added dropwise to 10 μ L afterwards with ultra-pure water, the DNA capture probe solution of 1 μM of biotin labeling, 4 DEG C be incubated 12h.
(5) electrode washing after incubation is totally added dropwise to 6 μ L, 1% BSA solution incubation at room temperature 30min afterwards with ultra-pure water.
(6) electrode cleaning buffer solution (the 10mM Na after above-mentioned BSA is closed2HPO4, 2mM KH2PO4, 37mM NaCl, 2.7mM KCl, pH 7.4) rinse well and dried in nitrogen.
(7) target dna of various concentrations is added dropwise and 37 DEG C of hybridization 2h is placed on electrode.
(8) 10 μ L detection probe mixed liquors are added dropwise on electrode after the drying and are placed in 37 DEG C of incubation 2h.
(9) it is placed in nitrogen and dries after the electrode after incubation is rinsed well with cleaning buffer solution.
(10) electrode is placed in 5mL, 0.1M PBS (0.1M Na2HPO4, 0.1M KH2PO4, 0.1M KCl) in carry out table Sign, 20 μ L, 1.4M H are added every 50s2O2, measure its chrono-amperometric variable-current value.
(11) linear according to gained current variation value and CYP2C19*2 gene DNA fragment concentration, drawing is bent Line.
Compared with prior art, a kind of electrochemical DNA biology of quantitative Diagnosis CYP2C19*2 allele of the invention passes The preparation method of sensor and application, its protrude the characteristics of be：
(1) c-C will be based on60-CeO2- PtNPs nano composite material is incorporated into electrochemical DNA biology as signal probe In the preparation of sensor, the catalytic performance of material is not only effectively raised, and improves the supported quantity of biomolecule, and then Improve sensitivity and the biocompatibility of electrochemical DNA biosensor；
(2) it is used to identify target dna by using the capture probe of biotin labeling, it not only ensures capture probe and existed The good orientation that the electrode surface of Avidin modification is fixed, and visited by increasing sensor surface for the capture of molecular recognition Pin density and then the sensitivity for improving sensor；
(3) electrochemical DNA biosensor prepared by this method can be that clopidogrel related gene (CYP2C19*2) provides New quantitative detecting method, it is intended to provide certain guidance for the personalized medicine of clopidogrel.
(4) identical nano material and method of modifying are used, using capture probe, signal probe and target dna Specific recognition, only the special of a variety of clopidogrel related gene heredity need to can be achieved by changing the nucleotide sequence of probe Property, highly sensitive detection in addition, the method is easy, quickly, is easy to implement commercialization, so as to promote the development of accurate medical science.
Brief description of the drawings：
Fig. 1 is the structure schematic diagram of the electrochemical DNA biosensor of the present invention.
Fig. 2 is the scanning electron microscope (SEM) photograph of the signal probe difference synthesis step of the present invention, EDS figures and XPS figures.
Fig. 3 is that the chrono-amperometric that the electrochemical DNA biosensor of the present invention obtains when detecting CYP2C19*2 genes becomes The linear relationship of galvanic current and concentration, and the specificity of sensor.
Embodiment：
The present invention is further elaborated with reference to specific embodiment, it should be appreciated that these embodiments are merely to illustrate The present invention rather than limitation the scope of the present invention.
Embodiment 1
Step 1.0.126g 2- amino terephthalic acid (TPA)s, 0.187g FeCl3·6H2O is dissolved in 15mL DMF, is mixed.Will Above-mentioned solution oil bath heating 4h, 200 μ L glacial acetic acid is added in 15min after heating starts, and heating controls solution drop after terminating Warm to room temperature.Resulting solution 10000/min is centrifuged 5 minutes, is respectively cleaned three times with DMF, absolute ethyl alcohol, ultra-pure water respectively.Gained Product vacuum is dispersed in formation 1mg mL in ultra-pure water again after drying 24h-1System, obtain Fe-MOFs；
Step 2.1mL HAuCl4(1%) 1mL (1mg mL are added-1) in Fe-MOFs solution, violent ultrasonic 15min.Gained Solution is placed on magnetic stirring apparatus, and under 400 revs/min of stirring, 2mL NaBH are added dropwise4(0.1M) is to solution by brown Discoloration is black, continues to stir 30min.Above-mentioned mixed solution 8000/min is centrifuged 5 minutes, is cleaned three times with ultra-pure water.Finally, The product of preparation is dried 12 hours at 50 DEG C, it is standby.
Step 3. is respectively with 0.3 and 0.05 μm of Al2O3Polishing electrode into minute surface, is then pressed ultra-pure water, nothing by powder respectively Order each 5min of ultrasound electrode of water-ethanol, ultra-pure water, drying at room temperature are standby；
Step 4. drips 6 μ L, the metal organic frame (AuNPs@Fe-MOFs) of electrode modified material golden nanometer particle@iron It is added in electrode surface, drying at room temperature；
Step 5. is with will be added dropwise 10 μ L, 100 μ g mL after pole drying-1Avidin solution is placed in 4 DEG C of incubation 12h；
Step 6. electrode washing after incubation is totally added dropwise afterwards with ultra-pure water 10 μ L, the DNA captures of 1 μM of biotin labeling Probe solution, 4 DEG C of incubation 12h；
Step 7. electrode washing after incubation is totally added dropwise afterwards with ultra-pure water 6 μ L, 1% BSA solution incubation at room temperature 30min；
Step 8. by above-mentioned BSA close after electrode cleaning buffer solution (10mM Na2HPO4, 2mM KH2PO4, 37mM NaCl, 2.7mM KCl, pH 7.4) rinse well and dried in nitrogen；
The target dna of various concentrations is added dropwise step 9. is placed in 37 DEG C of hybridization 2h on electrode；
10 μ L detection probe mixed liquors are added dropwise on the electrode of step 10. after the drying and are placed in 37 DEG C of incubation 2h；
Step 11. is placed in nitrogen after the electrode after incubation is rinsed well with cleaning buffer solution and dried；
Electrode is placed in 5mL, 0.1M PBS (0.1M Na by step 12.2HPO4, 0.1M KH2PO4, 0.1M KCl) in carry out Characterize, 20 μ L, 1.4M H are added every 50s2O2, measure its chrono-amperometric variable-current value；
Step 13. is linear according to gained current variation value and CYP2C19*2DNA fragment concentrations, and drawing is bent Line；Measurement result shows that CYP2C19*2 genetic fragment concentration is linear in the range of 1fM-50nM, and linearly dependent coefficient is 0.999, detection is limited to 0.33fM.
Step 14. is by the sensor of the present invention in 4 DEG C of preservations, discontinuity detection sensor current response, after storing 21 days Current-responsive is still the 90.4% of initial current, represents that sensor has good stability；
Step 15. present invention takes DNA biosensor 5 prepared by same batch, under the same conditions to 100pM's CYP2C19*2 gene DNA fragments are measured respectively, each determination of electrode 3 times, as a result the relative standard deviation of response current Less than 1.3%；Meanwhile the DNA biosensor 2 for taking different batches to prepare, under the same conditions to 100pM CYP2C19* 2 gene DNA fragments are measured respectively, each determination of electrode 3 times, and as a result the relative standard deviation of response current is less than 1.27%, illustrate in sensor batch and differences between batches are small, sensor reappearance is good.
The sensor of the present invention is used to detect target nucleic acid sequence by step 16., and base mismatch, as a result base mismatch is electric Stream response seems insignificant relative to target nucleic acid sequence, illustrates the specific good of sensor, can distinguish target sequence very well Row.
Described above is only the preferred embodiment of the present invention, it is noted that for the common skill of the art For art personnel, under the precondition for not departing from the principle of the invention, some improvements and modifications can also be made, these improve and Retouching also should be regarded as protection scope of the present invention.

Claims (3)

1. a kind of electrochemical sensor for CYP2C19*2 detections, it is characterised in that comprise the following steps：
(1) fullerene (c-C of carboxylated60)-ceria (CeO2)-nano platinum particle (PtNPs)-ssDNA detection probes Prepare；
(2) electrochemical DNA biosensor is established, determines CYP2C19*2 genes, draws standard curve.
2. c-C according to claim 160-CeO2The preparation process of-PtNPs-ssDNA compounds, its feature include following step Suddenly：
(1)c-C60-CeO2The preparation of nano composite material：
First, by 10mg c-C60It is dissolved in 5mL ultra-pure waters, is placed in and is ultrasonically treated 15min at room temperature, uniformly suspended Liquid.Then, by 10mL 0.025M Ce (NO3)3·6H2O mixes with 10mL 0.025M HTMA.Then two kinds of solution are mixed And be maintained in 80 DEG C of water-bath 5 hours, use ultra-pure water centrifuge washing 3 times after room temperature cooling.Then, by prepared sediment Dry at 60 DEG C, used for next step.Then, by 20mg c-C60/CeO2Compound is dispersed in 5mL ethanol, is ultrasonically treated 15 minutes, obtain dispersion soln.Then, 0.1mL APTES is mixed with above-mentioned solution, 1.5 is kept under the conditions of being placed in 70 DEG C Hour.After being cooled to room temperature, with ultra-pure water centrifuge washing, obtained c-C60/CeO2Complex.The product most prepared at last is 50 Dry at DEG C, further use.
(2)c-C60-CeO2The preparation of-PtNPs nano composite materials：
Pass through NaBH4Reducing process has synthesized c-C60/CeO2/PtNPs.First, to 1mL c-C60/CeO2Add in solution (2mg/mL) Enter 1mL H2PtCl6(1%), and it is ultrasonically treated 5 minutes.Then under agitation by 2mL NaBH4(0.1M) solution is added dropwise Into mixture, react 30 minutes, be then centrifuged for washing 3 times, and be dissolved in 1mL ultra-pure water and further use.
(3)c-C60-CeO2The preparation of-PtNPs-ssDNA compounds
By prepared 1mL c-C60/CeO2Signal probe (SP) amido modified with 200 μ L (1 μM)/PtNPs mixes, and 4 It is gently mixed at DEG C 24 hours.Then, after 1mg BSA are mixed with 1mL TE buffer solutions, it is added to c-C60/CeO2/ PtNPs is molten In liquid, continue to be gently mixed at 4 DEG C 4 hours, for blocking nonspecific binding site.Then, c- is centrifuged with 8000rpm C60/CeO2/ PtNPs/SP/BSA bioconjugates, washed 3 times with PBS, be dispersed in 1mL hybridization solutions and further use.
3. according to claim 1 establish electrochemical DNA biosensor, CYP2C19*2 genes are determined, it is bent to draw standard Line, it is characterised in that comprise the following steps：
(1) respectively with 0.3 and 0.05 μm of Al2O3Powder by polishing electrode into minute surface, then respectively by ultra-pure water, absolute ethyl alcohol, Order each 5min of ultrasound electrode of ultra-pure water, drying at room temperature are standby；
(2) metal organic frame (AuNPs@Fe-MOFs) of 6 μ L electrode modified material golden nanometer particle@iron is added dropwise in electrode Surface, drying at room temperature.
(3) by 10 μ L, 100 μ g mL-1Avidin is incorporated in dried electrode surface (4 DEG C, 12h).
(4) electrode washing is totally added dropwise to 10 μ L afterwards with ultra-pure water, the DNA capture probe solution of 1 μM of biotin labeling, 4 DEG C incubate Educate 12h.
(5) electrode washing after incubation is totally added dropwise to 6 μ L, 1% BSA solution incubation at room temperature 30min afterwards with ultra-pure water.
(6) electrode cleaning buffer solution (the 10mM Na after above-mentioned BSA is closed2HPO4, 2mM KH2PO4, 37mM NaCl, 2.7mM KCl, pH7.4) rinse well and dried in nitrogen.
(7) target dna of various concentrations is added dropwise and 37 DEG C of hybridization 2h is placed on electrode.
(8) 10 μ L detection probe mixed liquors are added dropwise on electrode after the drying and are placed in 37 DEG C of incubation 2h.
(9) it is placed in nitrogen and dries after the electrode after incubation is rinsed well with cleaning buffer solution.
(10) electrode is placed in 5mL, 0.1M PBS (0.1M Na2HPO4, 0.1M KH2PO4, 0.1M KCl) in characterized, often 20 μ L, 1.4M H are added every 50s2O2, measure its chrono-amperometric variable-current value.
(11) it is linear according to gained current variation value and CYP2C19*2 gene DNA fragment concentration, drawing curve.
CN201710803959.2A 2017-09-06 2017-09-06 A kind of electrochemical sensor for CYP2C19*2 detections Pending CN107723340A (en)

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CN112730547A (en) * 2020-12-28 2021-04-30 重庆医科大学 Preparation method and application of electrochemical biosensor for detecting NSCLC circulating tumor genes
CN114384133A (en) * 2022-01-14 2022-04-22 山东农业大学 Electrochemical sensor for detecting heavy metal lead ions in soil solution and construction method thereof

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Publication number Priority date Publication date Assignee Title
CN109207555A (en) * 2018-11-02 2019-01-15 南京工业大学 Colorimetric detection method for DNase I activity
CN109540983A (en) * 2019-01-22 2019-03-29 重庆医科大学 It is a kind of for detecting the novel electrochemical Biosensors of 2,6 sialylated glycan of α
CN109540983B (en) * 2019-01-22 2021-09-03 重庆医科大学 Novel electrochemical biosensor for detecting alpha 2,6 sialylated glycans
CN111926006A (en) * 2020-07-23 2020-11-13 国电科学技术研究院有限公司 Self-assembled label-free magnetic nano CYP2C9 x 3 gene probe and preparation method thereof
CN111926006B (en) * 2020-07-23 2024-03-19 国家能源集团科学技术研究院有限公司 Self-assembled label-free magnetic nano CYP2C9 x 3 gene probe and preparation method thereof
CN112730547A (en) * 2020-12-28 2021-04-30 重庆医科大学 Preparation method and application of electrochemical biosensor for detecting NSCLC circulating tumor genes
CN114384133A (en) * 2022-01-14 2022-04-22 山东农业大学 Electrochemical sensor for detecting heavy metal lead ions in soil solution and construction method thereof
CN114384133B (en) * 2022-01-14 2022-11-04 山东农业大学 Electrochemical sensor for detecting heavy metal lead ions in soil solution and construction method thereof

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