CN106979965A - One kind is used for hypertension individuation medication Gene A DRB1 1165C>The electrochemical sensor preparation method of G detections - Google Patents

One kind is used for hypertension individuation medication Gene A DRB1 1165C>The electrochemical sensor preparation method of G detections Download PDF

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CN106979965A
CN106979965A CN201710088007.7A CN201710088007A CN106979965A CN 106979965 A CN106979965 A CN 106979965A CN 201710088007 A CN201710088007 A CN 201710088007A CN 106979965 A CN106979965 A CN 106979965A
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mil
hemin
concentration
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solution
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袁国林
王磊
张吉才
毛达勇
王芳
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Shiyan Taihe Hospital
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Shiyan Taihe Hospital
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles

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Abstract

The present invention proposes a kind of for hypertension individuation medication Gene A DRB1 1165C>The electrochemical sensor preparation method of G detections, first using ferrous metal organic frame Fe MIL 88NH2Parcel ferroheme obtains Hemin@Fe MIL 88NH2Nano composite material, then nano platinum particle in-situ reducing make its functionalization that Pt/Hemin@Fe MIL 88NH are made in above-mentioned material surface2Composite, then adsorbs copper ion by the amino of composite material surface, Pt/Hemin Fe MIL 88NH is made2/Cu2+Composite.Using Pt/Hemin@Fe MIL 88NH2/Cu2+Composite uses it for electrode modification, immobilized capture probes, so as to be prepared for ADRB1 1165C>The electrochemical sensor of G genetic polymorphism detections, the sensor is successfully used in the detection that single base mutation occurs for ADRB1 genes.The advantage of the invention is that sensitivity is high, high specificity, detection is rapid, convenient.The present invention provides new detection method for metoprolol personalized medicine.

Description

One kind is used for hypertension individuation medication Gene A DRB1-1165C>The electrification of G detections Learn transducer production method
Technical field
The present invention relates to a kind of ADRB11165C for clinically instructing the hypertension personalized medicine such as Mei Tuonuoer>G bases Because of the preparation method and application of the electrochemical sensor of polymorphic detection, metal organic frame parcel ferroheme is based especially on Non-marked type biology sensor, for detecting ADRB11165C>G gene pleiomorphisms, belong to field of electrochemical detection.
Background technology
In the past decade, the change of identification disease specific gene causes targeted therapy to obtain fast development.In high blood In pressure treatment, carrying ADRB1 mutators by using metoprolol and its similar medicine targeting obtains its therapeutic effect Significantly improve, the other medicines of the therapeutic alliance with more high effect and less side effect are in clinical test, therefore, Patient is routinely screened and whether carries the mutation of the gene to determine whether they can benefit from such treatment. ADRB1 is β 1- adrenergic receptor genes, it is known that it is the target gene of hypertension beta-blocker.ADRB1 body cells are dashed forward Become, Arg389Gly（Rs1801253, c.1165C> G）It is present in 68.6-80.5% hypertension, Arg389Gly high blood Patient is pressed to need bigger dosage than carrying homozygosis Arg389Arg crowd, accordingly, it would be desirable to suitable for the spirit of the genotype detection Quick method.
The method of existing detection ADRB1 mutation depends on DNA sequencing and polymerase chain reaction（PCR）Deng.DNA sequencing is expensive Testing cost and slow detection cycle（2-3 weeks）It is set to cannot function as routine clinical detection means.PCR easily by Carry out the interference of biological sample complex component, while needing the pretreatment of sample and the nucleic acid purification mistake from bulk sample Journey.Therefore, in the urgent need to method that is a kind of more accurate and ADRB1 mutation can directly being detected in serum or blood.Closely Nian Lai, this omnipotent detection platform of electrochemical DNA biosensor is high due to its sensitivity, with low cost, quick and detection sample Measure the advantage such as few and obtain fast development in the context of detection of biomarker.
Ferroheme is a kind of well-known native metal porphyrin compound, with similar peroxidase sample activity.However, It is low that ferroheme usually faces catalytic activity as catalyst, the problems such as stability is poor in neutral and alkaline aqueous buffer solution.For These bottlenecks are broken through, development is urged to obtain as the supported matrix of ferroheme with strong catalytic activity and the bionical of high stability Agent is a kind of preferable mode.Fe-MIL-88NH2Metal organic frame is due to its high-specific surface area, adjustable aperture and exposure Avtive spot, be a kind of new host matrix material, available for the immobilized of ferroheme, development has high catalytic activity and length The solid catalyst of catalytic life.Ferroheme is wrapped in Fe-MIL-88NH by some researchers2Middle structure electrochemistry or chemistry hair Optical sensor is used for the detection of protein.
However, up to the present, Fe-MIL-88NH2Metal organic frame parcel ferroheme builds electrochemical DNA biosensor Seldom have been reported that.
The content of the invention
It is used for hypertension individuation medication Gene A DRB1-1165C it is an object of the invention to provide one kind>The electrification of G detections Transducer production method is learned, for by using Pt/Hemin@Fe-MIL-88NH2/Cu2 +It is used as amplification of signal platform Super quick detection ADRB1 mutators, nano platinum particle, hemin, Cu2+Synergy can not only increase to H2O2's Electro catalytic activity, and effective fixation of capture probe can be improved, detection range is greatly expanded, while it is clinical real to demonstrate it The feasibility of border application.
To achieve the goal, the present invention is adopted the following technical scheme that：
One kind is used for hypertension individuation medication Gene A DRB1-1165C>The electrochemical sensor preparation method of G detections, it is special Levy is to comprise the following steps：
(1) nano platinum particle/ferroheme@metal organic frames/copper ion（Pt/Hemin@Fe-MIL-88NH2/Cu2+）Nanometer The preparation of composite；
(2) using Pt/Hemin@Fe-MIL-88NH2/Cu2 +As nano composite material amplification of signal platform, electrification is set up Learn DNA biosensor；
(3) applying step（2）Obtained electrochemical DNA biosensor is to Gene A DRB1-1165C>G is detected.
In the present invention, above-mentioned steps（1）" nano platinum particle/ferroheme@metal organic frames/copper ion（Pt/Hemin@ Fe-MIL-88NH2/Cu2+）The preparation of nano composite material；", comprise the following steps：
(1.1) Hemin@Fe-MIL-88NH2The preparation of nano composite material：
0.187g ferric chloride (FeCl36H2O)（FeCl3•6H2O）, 0.226g ferroheme and 0.126g diaminourea be to benzene two Formic acid is dissolved in 15 mL dimethyl sulfoxide (DMSO)s（DMF）In, mixed solution is placed in 120 DEG C of oil bath 4h in silicones；Then 3.45 are added Mmol ascorbic acid, heats 15min；Room temperature is gradually cooling to, is centrifuged, it is clear successively with dimethyl sulfoxide (DMSO), ethanol, deionized water Wash, gained nano material sediment, which is placed in vacuum drying chamber, after centrifugation is dried overnight, and obtains the blood of metal organic frame parcel Red pigment nano composite material Hemin@Fe-MIL-88NH2/Cu2 +；
(1.2) Pt/Hemin@Fe-MIL-88NH2The preparation of nano composite material：
The Hemin@Fe-MIL-88NH of nano platinum particle functionalization are synthesized by using the method for sodium borohydride reduction2；First, With the chloroplatinic acid (H of 1mL mass percent concentrations 1%2PtCl6) it is added to the Hemin@Fe-MIL- that 1mL concentration is 1mg/mL 88NH2In solution, ultrasonic 20 min；Then the sodium borohydride (NaBH that 2 mL concentration are 0.1M4) solution be added dropwise it is above-mentioned mixed Close in solution, 400 rpm stir 30 min；Mixed solution is centrifuged, and is washed three times, and nano platinum particle is connected by Pt-N keys and combined In Hemin@Fe-MIL-88NH2Surface, obtains Pt/Hemin@Fe-MIL-88NH2Nano composite material；
(1.3) Pt/Hemin@Fe-MIL-88NH2/Cu2+The preparation of composite：
First, the Pt/Hemin@Fe-MIL-88NH that 1 mL concentration is 2mg/mL2Nanocomposite solution is scattered in 10 ML concentration is 2mg/mL copper nitrate（Cu(NO3)2）In the aqueous solution, 24h is stirred at room temperature, makes copper absorption in Pt/Hemin@ Fe-MIL-88NH2Nano composite material surface；Gained heterocomplex is centrifuged, and washes 3 times to remove uncombined Cu2+, obtain Pt/ Hemin@Fe-MIL-88NH2/Cu2+Nano composite material.
In the present invention, above-mentioned steps（2）" use Pt/Hemin@Fe-MIL-88NH2/Cu2+Believe as nano composite material Number amplification platform, sets up electrochemical DNA biosensor ", it is characterised in that include following steps：
(2.1) 0.3mm and 0.05mm aluminum oxide is used successively respectively（Al2O3）Powder into minute surface, then distinguishes polishing electrode By ultra-pure water, absolute ethyl alcohol, ultra-pure water ultrasonic each 5 min of electrode of order, drying at room temperature is standby；
(2.2) by Pt/Hemin@Fe-MIL-88NH of the 6 mL concentration for 2 mg/mL2/Cu2+Nanocomposite solution is added dropwise In electrode surface, drying at room temperature；
(2.3) it is the SEQ ID NO in 1 μM of sequence table by 10 mL concentration：The solution of DNA fragmentation capture probe shown in 1 is added dropwise to Electrode surface, 4 DEG C of 12 h of incubation；
(2.4) with cleaning buffer solution by the electrode washing after incubation it is clean after, 6 μ L concentration of dropwise addition be 1.0mM hexyl mercaptans（HT）Room Temperature is incubated 30 min, obtains electrochemical DNA biosensor；Wherein, the component composition of the cleaning buffer solution：Concentration is 10 MM Na2HPO4, concentration be 2 mM KH2PO4, the KCl that concentration is 37 mM NaCl, concentration is 2.7 mM, remaining be Ionized water；PH is 7.4.
In the present invention, above-mentioned steps（3）" Applied Electrochemistry DNA biosensor is to Gene A DRB1-1165C>G is examined Survey ", it is characterised in that comprise the following steps：
(3.1) with cleaning buffer solution by the electrode washing after incubation it is clean after by concentration successively from 1fM, 10fM, 100fM, 1pM, 10pM target DNA fragments solution（The SEQ ID NO that the DNA is shown in sequence table：Shown in 2）It is added dropwise in same batch The Different electrodes surface of structure, 37 DEG C of hybridization 2h；
(3.2) it is placed in nitrogen and dries after the electrode after incubation is rinsed well with cleaning buffer solution；
(3.3) electrode is placed in 7 mL phosphate buffer solutions and characterized, after treating that background current is stable, 20 μ L, 1.2 mol L-1 H2O2 is added in above-mentioned phosphate buffer solution, its electric current of i-t curve records under -0.7 V current potentials Value；Wherein, the component composition of the phosphate buffer solution：Concentration is 0.1M Na2HPO4, concentration be 0.1M KH2PO4, it is dense Spend the KCl for 0.1 M, remaining be deionized water；
(3.4) according to gained peak current and target DNA fragments solution（The SEQ ID NO that the DNA is shown in sequence table：Shown in 2） Concentration is linear, draws standard curve.
Beneficial effect：Compared with prior art, it is the characteristics of present invention protrusion：
(1) in order to improve the test limit of sensor, the nano platinum particle with good biocompatibility is introduced（PtNPs）For Concerted catalysis H2O2.Due to platinum/ferroheme@Fe-MIL-88NH2The big surface area of nano composite material, high conductivity and excellent Adsorption capacity, its surface amino groups functionalization can be used for Cu2 +Absorption, can further promote the redox of hydrogen peroxide.It is based on Pt /Hemin@ Fe-MIL-88NH2 / Cu2+Cooperative effect, obtain with multiple signal amplify；By nano platinum particle, Ferroheme, copper ion is incorporated into the preparation of electrochemical DNA biosensor, is constructed with multiple cascade catalyzed signal amplification The nano composite material of performance is modified for sensing interface, not only increases the supported quantity of biomolecule, and improve electrification Learn sensitivity and the biocompatibility of DNA biosensor；
(2) this method is detected to ADRB1 genes, it is not necessary to sample nucleic is marked and PCR enhanced processings；
(3) electrochemical DNA biosensor prepared by this method can adjust metoprolol to be clinical according to different patient's genotype Dosage；
(4) identical nano material and method of modifying are used, using capture probe, signal probe and target dna it is special Property identification, only the special of a variety of disease (such as tumour) personalized medicine genes 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 translational medicine.
Brief description of the drawings
Fig. 1 is the structure schematic diagram of the electrochemical DNA biosensor of the present invention.
Fig. 2 is Pt/Hemin@Fe-MIL-88NH of the invention2Field emission scanning electron microscope figure, the energy of nano composite material Diffraction photoelectron spectroscopy figure and X-ray photoelectron spectroscopic analysis figure.
Fig. 3 is detecting ADRB1-1165C for the electrochemical DNA biosensor of the present invention>Obtained during G gene pleiomorphisms The linear relationship of i-t curves and concentration, and sensor specificity and reappearance.
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
With reference to shown in Fig. 1, embodiments of the present invention are further described as follows：
Step 1. 0.187g ferric chloride (FeCl36H2O)（FeCl3•6H2O）, 0.226g ferroheme and 0.126g diaminourea pair Phthalic acid is dissolved in 15 mL dimethyl sulfoxide (DMSO)s（DMF）In, mixed solution is placed in 120 DEG C of oil bath 4h in silicones；Then 3.45 Mmol ascorbic acid is added in above-mentioned solution, heats 15min；Be gradually cooling to room temperature, centrifuge, DMF, ethanol, deionized water according to Secondary cleaning, sediment is placed in vacuum drying chamber and is dried overnight, and obtains the ferroheme of metal organic frame parcel（Hemin@Fe- MIL-88NH2）Nano composite material；
Hemin@Fe-MIL-88NH of the step 2. in synthetic 1 mL2（1mg/mL）1mL mass percents are added in solution dense Spend the chloroplatinic acid (H for 1%2PtCl6), violent ultrasonic 20 min；Then it is 0.1M sodium borohydrides (NaBH 2 mL concentration4) molten Liquid is added dropwise in above-mentioned mixed solution, and 400 rpm stir 30 min；Mixed solution is centrifuged, and is washed three times, nano platinum particle can Hemin@Fe-MIL-88NH are incorporated into by the connection of Pt-N keys2Surface, obtains the Hemin@Fe- of nano platinum particle functionalization MIL-88NH2（Pt/Hemin@Fe-MIL-88NH2）；
Step 3. is by 1 mL, 2 mg/mL Pt/Hemin@Fe-MIL-88NH2It is scattered in 10 mL, 2 mg/mL Cu (NO3)2Water In solution, 24 h are stirred at room temperature, make copper absorption in Pt/Hemin@Fe-MIL-88NH2Surface；Gained heterocomplex is centrifuged, water Wash repeatedly, obtain Pt/Hemin@Fe-MIL-88NH2/Cu2+Nano composite material；
From Fig. 2A, 2B Pt/Hemin@Fe-MIL-88NH2The visible many bright spot spline structures of field emission scanning electron microscope figure be distributed in Hemin@Fe-MIL-88NH2Surface, this is nano platinum particle, shows that nano platinum particle has successfully been modified in Hemin@Fe-MIL- 88NH2Surface；Fig. 2 C are Pt/Hemin@Fe-MIL-88NH2Energy diffraction photoelectron spectroscopy figure, from scheme it is visible except C, N, Clearly visible Pt elements peak beyond O, Fe element peak, again shows that nano platinum particle has successfully been modified successfully in Hemin Fe- MIL-88NH2Surface；Fig. 2 D, 2E, 2E are Pt/Hemin@Fe-MIL-88NH2X-ray photoelectron spectroscopic analysis figure, from figure It can be seen that Pt4f, C1s, N1s, O1s and Fe2p characteristic peak, further illustrate Pt/Hemin@Fe-MIL-88NH2Successfully close Into；
Step 4. uses 0.3mm and 0.05mm aluminum oxide respectively（Al2O3）Powder into minute surface, then presses polishing electrode respectively Ultra-pure water, absolute ethyl alcohol, order each 5 min of ultrasonic electrode of ultra-pure water, drying at room temperature are standby；
6 mL concentration are 2mg/mLPt/Hemin@Fe-MIL-88NH by step 5.2/Cu2+It is added dropwise in electrode surface, room temperature is done It is dry；
10 mL concentration are the SEQ ID NO in 1 μM of sequence table by step 6.：The solution of DNA fragmentation capture probe shown in 1 is added dropwise To electrode surface, 4 DEG C of 12 h of incubation；
Step 7. electrode washing after incubation is totally added dropwise afterwards with cleaning buffer solution 6 μ L concentration for 1.0 mM hexyl mercaptans（HT） 30 min are incubated at room temperature, electrochemical DNA biosensor is obtained；Wherein, the component composition of the cleaning buffer solution：Concentration is 10 MM Na2HPO4, concentration be 2mM KH2PO4, concentration be 37mM NaCl, concentration be 2.7mM KCl, remaining be deionization Water；PH is 7.4；
Step 8. is by the sensor of the present invention in 4 DEG C of preservations, the response of discontinuity detection sensor current, electric current after storing 28 days Response is still the 91.17% of initial current, and surface probe has good stability；The present invention takes prepared by same batch DNA biosensor 5, under the same conditions to 0.1,10 and 1000fM ADRB1-1165C>G gene DNA fragments are distinguished It is measured, each concentration mensuration 5 times, as a result the relative standard deviation of response current is less than 4.11%；Meanwhile, take different batches The DNA biosensor of preparation 3, under the same conditions to 0.1,10 and 1000fM ADRB1-1165C>G gene DNA pieces Section is measured respectively, each concentration mensuration 5 times, and as a result the relative standard deviation of response current is less than 4.33%, illustrates sensor Batch in and differences between batches it is small, sensor reappearance is good；
Step 9. with cleaning buffer solution by the electrode washing after incubation it is clean after by various concentrations（Concentration be followed successively by 1fM, 10fM, 100 fM、1pM、10pM）Target DNA fragments solution（The SEQ ID NO that the DNA fragmentation is shown in sequence table：Shown in 2）It is added dropwise In electrode surface, 37 DEG C of hybridization 2h；
Step 10 is placed in nitrogen after the electrode after incubation is rinsed well with cleaning buffer solution and dried；
Electrode is placed in 7 mL phosphate buffer solutions and characterized by step 11., after treating that background current is stable, adds 20 μ L concentration is 1.2 mol/L H2O2Add in above-mentioned phosphate buffer solution, its electricity of i-t curve records under 0.7 V current potentials Flow valuve；Wherein, the component composition of the phosphate buffer solution：Concentration is 0.1M Na2HPO4, concentration be 0.1M KH2PO4、 Concentration for 0.1 M KCl, remaining be deionized water；
Step 12. is according to gained peak current and ADRB1-1165C>The DNA fragmentation solution of G genes（The DNA is shown in sequence table SEQ ID NO：Shown in 2）Concentration is linear, drawing curve；Measurement result shows ADRB1-1165C>G genes DNA fragmentation concentration is linear in the range of 1fM-10nM, and linearly dependent coefficient is 0.998, detects spacing 0.33 fM；
The sensor of the present invention is used to detect target nucleic acid sequence by step 13.（SEQ ID NO in sequence table：2 institutes Show）, base mismatch（SEQ ID NO in sequence table：3rd, shown in 4,5）And random sequence（SEQ ID NO in sequence table：6 institutes Show）, as a result base mismatch current-responsive seem insignificant relative to target nucleic acid sequence, illustrate the specific good of sensor, Target sequence can be distinguished very well and can exclude blank plasma matrix interference；
Reduce from the current-responsive value of Fig. 3 A visible light transducers with the increase of target gene concentration to be measured, and have well Linear dependence（Fig. 3 B）；From 3C visible light transducers to ADRB1-1165C>G target genes（ADRB1）When being detected, electric current Response is far longer than single base mismatch（SBM）, double alkali yl mispairing（DBM）, three base mispairings（TBM）, random sequence （Random）And blank plasma（Blank）, illustrate that the selectivity of sensor is good；From in Fig. 3 D, sensor batch （intraday）, batch between（interday）Difference very little, illustrates that the reappearance of sensor is good, can be advantageously used in target substance point Analysis.
From figure 3, it can be seen that ringing I to the measurement electric current of blank plasma with sensor0It should be worth for baseline, then using sensing Device is detected to the test plasma containing genetic fragment；If containing ADRB1-1165C in test plasma>G target DNA fragments, The then current-responsive value I of sensor1Current-responsive value I of the meeting compared with blank plasma0It is significantly raised, and I0And I1Two current-responsives There can be obvious significant difference between value；If not containing ADRB1-1165C in test plasma>G target sequences（It is probably now single Other materials in base mispairing, double alkali yl mispairing, three base mispairings, random sequence or blood plasma）, then the current-responsive value of sensor I1Meeting and the current-responsive value I of blank plasma0Without significant change, and I0And I1Statistics is not present between two current-responsive values poor It is different.Electrochemical sensor intuitive measurement results as shown in Figure 3 C, i.e. ADRB1-1165C>G patient needs bigger than unmutated crowd Dosage.
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.
In the present invention, the implication of described concentration symbology is as follows：
M：mol/L（Mol/L）；
mM：mmol/L（MM/l）；
μM：μmol/L（Micromoles per liter）；
nM：nmol/ L（Nanomole/liter）；
fM：fmol/ L（Femtomole/liter）；
pM：pmol/ L（Picomole/liter）.
Sequence table
<110>Taihe Hospital, Shiyan
<120>One kind is used for hypertension individuation medication Gene A DRB1-1165C>The electrochemical sensor preparation method of G detections
<160> 6
<210> 1
<211> 12
<212> DNA
<213>Artificial sequence
<220>
<223>Designed according to size and polarity, capture probe is combined as use as with biotin
<400> 1
gaagtggcag ca 12
<210> 2
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>Designed according to size and polarity, for use as target-gene sequence
<400> 2
tgctgccact tcgtcaccaa ccgggcctac 30
<210> 3
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>Designed according to size and polarity, for use as random sequence
<400> 3
catccatcgt atgtcaccaa ccgggcctac 30
<210> 4
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>Designed according to size and polarity, for use as the single base mismatch sequence corresponding with target gene
<400> 4
tgctgcgact tcgtcaccaa ccgggcctac 30
<210> 5
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>Designed according to size and polarity, for use as the double alkali yl mismatch corresponding with target gene
<400> 5
tcctgcgact tcgtcaccaa ccgggcctac 30
<210> 6
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>Designed according to size and polarity, for use as the three base mispairing sequences corresponding with target gene
<400> 6
tcctgcgact tggtcaccaa ccgggcctac 30

Claims (3)

1. one kind is used for hypertension individuation medication Gene A DRB1-1165C>The electrochemical sensor preparation method of G detections, its It is characterized in comprise the following steps：
(1) nano platinum particle/ferroheme@metal organic frames/copper ion（Pt/Hemin@Fe-MIL-88NH2/Cu2+）Nanometer The preparation of composite；
(2) Pt/Hemin@Fe-MIL-88NH are made2/Cu2 +As nano composite material amplification of signal platform, electrochemical DNA is set up Biology sensor.
2. a kind of according to claim 1 be used for hypertension individuation medication Gene A DRB1-1165C>The electrochemistry of G detections Transducer production method, it is characterized in that, the step（1）" nano platinum particle/ferroheme@metal organic frames/copper ion（Pt/ Hemin@Fe-MIL-88NH2/Cu2+）The preparation of nano composite material；", comprise the following steps：
(1.1) Hemin@Fe-MIL-88NH2The preparation of nano composite material：
The ferroheme of ferric chloride (FeCl36H2O), 0.226g 0.187g and 0.126g diaminourea terephthalic acid (TPA) are dissolved in 15mL bis- In methyl sulfoxide, mixed solution is placed in 120 DEG C of oil bath 4h in silicones；Then 3.45mmol ascorbic acid, heating are added 15min；Room temperature is gradually cooling to, centrifuges, is cleaned successively with dimethyl sulfoxide (DMSO), ethanol, deionized water, gained nanometer material after centrifugation Material sediment, which is placed in vacuum drying chamber, to be dried overnight, and obtains the ferroheme nano composite material of metal organic frame parcel Hemin @ Fe-MIL-88NH2/Cu2 +；
(1.2) Pt/Hemin@Fe-MIL-88NH2The preparation of nano composite material：
The Hemin@Fe-MIL-88NH of nano platinum particle functionalization are synthesized by using the method for sodium borohydride reduction2；First, use The chloroplatinic acid of 1mL mass percent concentrations 1% is added to the Hemin@Fe-MIL-88NH that 1mL concentration is 1mg/mL2In solution, 20 min of ultrasound；Then 2 mL concentration are added dropwise in above-mentioned mixed solution for 0.1M sodium borohydride solution, 400 rpm are stirred Mix 30 min；Mixed solution is centrifuged, and is washed three times, and nano platinum particle can be incorporated into Hemin@Fe-MIL- by the connection of Pt-N keys 88NH2Surface, obtains Pt/Hemin@Fe-MIL-88NH2Nano composite material；
(1.3) Pt/Hemin@Fe-MIL-88NH2/Cu2+The preparation of composite：
First, the Pt/Hemin@Fe-MIL-88NH that 1mL concentration is 2 mg/mL2Nanocomposite solution is scattered in 10 mL Concentration for 2mg/mL copper nitrate aqueous solution in, 24h is stirred at room temperature, makes copper absorption in Pt/Hemin@Fe-MIL-88NH2 Nano composite material surface；Gained heterocomplex is centrifuged, and washes 3 times to remove uncombined Cu2+, obtain Pt/Hemin@Fe-MIL- 88NH2/Cu2+Nano composite material.
3. a kind of according to claim 1 be used for hypertension individuation medication Gene A DRB1-1165C>The electrochemistry of G detections Transducer production method, it is characterized in that, the step（2）" use Pt/Hemin@Fe-MIL-88NH2/Cu2+It is multiple as nanometer Condensation material amplification of signal platform, sets up electrochemical DNA biosensor ", comprise the following steps：
(2.1) distinguish polishing electrode successively with 0.3mm and 0.05mm alumina powder into minute surface, then respectively by ultrapure Water, absolute ethyl alcohol, order each 5 min of ultrasonic electrode of ultra-pure water, drying at room temperature are standby；
(2.2) by Pt/Hemin@Fe-MIL-88NH of the 6mL concentration for 2mg/mL2/Cu2+Nanocomposite solution is added dropwise Electrode surface, drying at room temperature；
(2.3) it is the SEQ ID NO in 1 μM of sequence table by 10 mL concentration：The solution of DNA fragmentation capture probe shown in 1 is added dropwise to Electrode surface, 4 DEG C of 12 h of incubation；
(2.4) with cleaning buffer solution by the electrode washing after incubation it is clean after, 6 μ L concentration of dropwise addition be 1.0mM hexyl mercaptans（HT）Room Temperature is incubated 30 min, obtains electrochemical DNA biosensor.
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CN107694604A (en) * 2017-08-23 2018-02-16 吉林大学 A kind of secondary heme/metal organic framework composite, preparation method and its application in phenol degrading
CN108195920A (en) * 2018-01-31 2018-06-22 大丰跃龙化学有限公司 For detecting the preparation of the electrochemical sensor of 2,4,6- trichlorophenol, 2,4,6,-Ts and analysis method
CN109182480A (en) * 2018-09-05 2019-01-11 华中科技大学 Application of the metal-organic framework material in polymerase chain reaction
CN109374709A (en) * 2018-10-23 2019-02-22 扬州大学 Based on metal-organic framework material-ferroheme electrochemical sensor preparation method and its usage
CN110672575A (en) * 2019-11-06 2020-01-10 湖北师范大学 Be used for detecting Hg2+And Cu2+Ratiometric fluorescent sensor, and preparation method and application thereof
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107694604A (en) * 2017-08-23 2018-02-16 吉林大学 A kind of secondary heme/metal organic framework composite, preparation method and its application in phenol degrading
CN108195920A (en) * 2018-01-31 2018-06-22 大丰跃龙化学有限公司 For detecting the preparation of the electrochemical sensor of 2,4,6- trichlorophenol, 2,4,6,-Ts and analysis method
CN109182480A (en) * 2018-09-05 2019-01-11 华中科技大学 Application of the metal-organic framework material in polymerase chain reaction
CN109374709A (en) * 2018-10-23 2019-02-22 扬州大学 Based on metal-organic framework material-ferroheme electrochemical sensor preparation method and its usage
CN110672575A (en) * 2019-11-06 2020-01-10 湖北师范大学 Be used for detecting Hg2+And Cu2+Ratiometric fluorescent sensor, and preparation method and application thereof
CN110672575B (en) * 2019-11-06 2021-12-07 湖北师范大学 Be used for detecting Hg2+And Cu2+Ratiometric fluorescent sensor, and preparation method and application thereof
CN114990197A (en) * 2022-06-27 2022-09-02 军事科学院军事医学研究院环境医学与作业医学研究所 Colorimetric sensing system for detecting kanamycin
CN114990197B (en) * 2022-06-27 2023-10-24 军事科学院军事医学研究院环境医学与作业医学研究所 Colorimetric sensing system for detecting kanamycin

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