CN103743805A - Biosensor based on nitrogen-hybridized mesoporous carbon as well as preparation method and application of biosensor - Google Patents
Biosensor based on nitrogen-hybridized mesoporous carbon as well as preparation method and application of biosensor Download PDFInfo
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Abstract
The invention relates to a biosensor based on nitrogen-hybridized mesoporous carbon as well as a preparation method and application of the biosensor. The biosensor comprises a working electrode, wherein the working electrode comprises a glassy carbon electrode; the surface of the detecting end of the glassy carbon electrode is sequentially connected with an L-cysteine, nitrogen-hybridized mesoporous carbon, a gold nanoparticle film and a sulfydryl-modified capture probe; the gold nanoparticle film is also connected with mercaptoethannol. The invention also provides an application of the biosensor. The application comprises the following steps: dropwise adding an object chain contained liquid to be detected and a signal probe contained liquid on the surface of the working electrode; dropwise adding a nanogold cluster labeled horse radish peroxidase-streptavidin contained buffer solution; and measuring in an electrolytic tank with hydroquinone and hydrogen peroxide as substrates, wherein the electrolytic tank is connected with a three-electrode system. The biosensor is high in sensitivity, rapid in response, high in detecting precision, relatively strong in anti-interference, simple and convenient in application operation, high in efficiency and low in detecting cost.
Description
Technical field
The present invention relates to a kind of biology sensor, specifically relate to a kind of biology sensor, preparation method and application thereof based on azepine mesoporous carbon.
Background technology
In terrestrial ecosystems, lignin is the aromaticity superpolymer that contains oxo phenylpropanol or derivatives thereof structural unit in a kind of amorphous, molecular structure being extensively present in plant, very common in agricultural and domestic waste, and the content in xylophyta accounts for 25%, be to be only second in the world the abundantest organism of cellulosic second.The natural degradation speed of lignin is very slow, conventionally adopts the method for piling up or burning to process, and not only can produce such as CO
x, a large amount of harmful gases such as methane, cause the wasting of resources, thereby Biodegradation of Lignin is more helpful for environmental resource simultaneously.White-rot fungi has biodegrability to lignin.White-rot fungi can secrete some ectoenzyme such as the oxidation such as laccase, manganese peroxidase, LiP lignin degradings, wherein especially the most key with the effect of manganese peroxidase (MnP).Therefore, detect the feature coding gene of MnP, can effectively understand the dynamic change of the MnP secreting in fungal organism lignin degrading process, thereby can control more accurately and effectively whole biodegradation process.
Adopting biosensor technique to detect genetic fragment is a trend of gene analysis test, and wherein electrochemical sensor is paid close attention to widely, because it possesses response fast, highly sensitive, high selectivity and the advantage such as workable.For DNA sensor, common working electrode is gold electrode or the screen printing electrode based on nm of gold, because the gene probe that is modified with sulfydryl is by sulfydryl and golden being cross-linked, is easy to be assemblied in electrode surface, carries out coherent detection.But, gold electrode is expensive, and screen printing electrode is disposable electrode, and for a large amount of detections, its total cost is also very considerable.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, provide a kind of highly sensitive, quick response, high measurement accuracy and compared with the biology sensor based on azepine mesoporous carbon of strong anti-interference, the present invention also provides a kind of preparation method of the simple biology sensor based on azepine mesoporous carbon, the present invention also provides the application of a kind of biology sensor based on azepine mesoporous carbon aspect manganese peroxidase specific coding genetic fragment measurement of concetration, described application operating is easy, efficiently, measurement cost is low.
The technical scheme that the present invention proposes is,
A kind of biology sensor based on azepine mesoporous carbon, comprise working electrode, described working electrode comprises glass-carbon electrode, the surface, test side of described glass-carbon electrode is connected with the capture probe of Cys, azepine mesoporous carbon, Au nanoparticles films and sulfydryl modification in turn, is also connected with the mercaptoethanol of sealing Au nanoparticles films residue binding site on described Au nanoparticles films.
Biology sensor based on azepine mesoporous carbon of the present invention is preferably three-electrode electro Chemical sensor, comprises working electrode, contrast electrode and to electrode, described contrast electrode is preferably saturated calomel electrode, described electrode is preferably to platinum electrode.
The test side surface deposition of described glass-carbon electrode of the present invention has Cys, Cys is connected with azepine mesoporous carbon, azepine mesoporous carbon outside is connected with Au nanoparticles films, described Au nanoparticles films is connected with the capture probe of sulfydryl modification, on described Au nanoparticles films, be also connected with the mercaptoethanol of sealing Au nanoparticles films residue binding site, make golden nanometer particle not with the combination such as signal probe or manganese peroxidase specific coding genetic fragment.
The capture probe of described sulfydryl modification is: 5'-HS-(CH
2)
6-T
1-3', T
1base sequence (being SEQ ID NO.1) be 5'-CTGATGGTGTCGTGTTTCT-3'.
The preparation method of described azepine mesoporous carbon is, by mesoporous silicon material, phenixin, ethylenediamine mixes, mesoporous silicon material is preferably mesoporous silicon template SBA-15, mesoporous silicon material, phenixin, the weight ratio of ethylenediamine is preferably 0.5～1.5:3:1.35, at 90～100 ℃, add thermal agitation, time is preferably 6～10h, condensing reflux, time is preferably 6～8h, be preferable under 40～60 ℃ of conditions dry, be placed in nitrogen, or in the mixed gas of nitrogen and hydrogen, be heated to 600～900 ℃ of processing, the preferred process time is 5～7h, preferred type of heating is that control heating rate is 3～5 ℃/min.After being disposed, add hydrofluorite, the massfraction of described hydrofluorite is preferably 5～7%, filter, and washing, dry, baking temperature is preferably 40～60 ℃, obtains azepine mesoporous carbon.
Mesoporous silicon template SBA-15 can obtain by being prepared as follows method, segmented copolymer P123 is placed in to hydrochloric acid and dissolves, and then dropwise adds ethyl orthosilicate, the mass ratio of segmented copolymer P123 and ethyl orthosilicate is 8: 17～23, and temperature is controlled at 30～35 ℃, stirs, potpourri, then by described potpourri 140～150 ℃ of heating, after reacting completely, be generally 23～25h, suction filtration, washing is to neutral, air-dry, 530～550 ℃ of roastings, obtain mesoporous silicon template SBA-15 again.
The present invention also provides a kind of preparation method of the biology sensor based on azepine mesoporous carbon, the preparation method of described working electrode is, on glass-carbon electrode, by electrochemical process, deposit Cys, then add azepine mesoporous carbon suspending liquid, last electrochemical process deposited gold nano particle, obtains modified electrode; The capture probe of sulfydryl modification is joined on described modified electrode, then add mercaptoethanol, clean, obtain described working electrode.
The spreading agent of described azepine mesoporous carbon suspending liquid is DMF or ultrapure water.
The present invention also provides a kind of described biology sensor based on azepine mesoporous carbon to measure the application of manganese peroxidase specific coding genetic fragment concentration, its step is, the liquid to be measured that contains object chain and the fluid drips that contains signal probe are added in to the working electrode surface of the described biology sensor based on azepine mesoporous carbon, after having reacted, the damping fluid that dropping contains nano golden cluster mark horseradish peroxidase-streptavidin (GNCs-HRP-SA), last in being connected to the electrolytic cell of three-electrode system, take p-dihydroxy-benzene and hydrogen peroxide as substrate, measure, according to equation of linear regression, obtain the concentration of object chain in liquid to be measured, described equation of linear regression is:
Y=(7.527±0.1787)X+(-66.5603±2.641)
Wherein, Y is current average; X is the natural logarithm of object chain concentration, and unit is molL
-1; The range of linearity of object chain concentration is 1 * 10
-19～1 * 10
-10m, measurement lower limit is 2 * 10
-20m.
Described signal probe is: 5'-T
2-biotin-3', T
2base sequence (being SEQ ID NO.2) be 5'-GATGCCGTTGTTGGCGGAGAA-3.
The base sequence of object chain in described liquid to be measured (being SEQ ID NO.3), i.e. manganese peroxidase specific coding genetic fragment is:
5'-TTCTCCGCCAACAACGGCATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGAAACACGACACCATCAG-3'。
The preparation method of described nano golden cluster mark horseradish peroxidase-streptavidin (GNCs-HRP-SA) is, chlorauric acid solution and horseradish peroxidase-streptavidin solution are mixed, under stirring condition, drip ascorbic acid solution, solution adds strong base solution after becoming muddiness, described strong base solution is preferably sodium hydroxide solution, stirs, standing, dialysis, obtains GNCs-HRP-SA.The quality proportion optimization of described gold chloride, horseradish peroxidase-streptavidin, ascorbic acid is 50～100:4～9:1～1.5, most preferably is 50:4.2:1.05.
The described damping fluid that contains nano golden cluster mark horseradish peroxidase-streptavidin is phosphate buffered solution, and pH is preferably 6.5～7.5, most preferably is 7.38.
In recent years, along with novel nano-material and the swift and violent combination of electrochemical sensing technology, in sensor construction strategy, in conjunction with new material, learn relevant feature, use some nano materials and biomolecule material to form structure of composite membrane and be modified at glass-carbon electrode surface.Nano material, because have high electronic conductivity, can provide bigger serface such as golden nanometer particle etc., keeps the advantages such as biologically active, is considered to outstanding biomolecule carrier and signal and transmits medium, can improve the sensitivity of electrochemical sensor.
Compared with prior art, the invention has the advantages that: azepine mesoporous carbon has good affinity to biomolecule, biocompatibility, and in azepine mesoporous carbon, there is π-π * electronic switch, thus improve the ability that passes to of electronics.Cys is nontoxic, biological fitness is strong, have film forming ability.
Nano golden cluster mark horseradish peroxidase-streptavidin amplifies material as signal, because nano golden cluster core itself has the ability of catalysis and conduction electron, horseradish peroxidase-the streptavidin around wrapping up at it has catalytic action equally, therefore, nano golden cluster mark horseradish peroxidase-streptavidin has dual signal expansion effect, and then improves the sensitivity of sensor.
The present invention has taken into full account azepine mesoporous carbon, nm of gold, Cys, nano golden cluster mark horseradish peroxidase-streptavidin character separately, and utilize the composite membrane that their form, build highly sensitive, quick response, high measurement accuracy and compared with the biology sensor based on azepine mesoporous carbon of strong anti-interference, the described biology sensor based on azepine mesoporous carbon is for detection of MnP genetic fragment, easy and simple to handle, efficiently, testing cost is low, for monitoring and the control procedure of microbial degradation lignin provides a kind of effective biology tool.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope (SEM) photograph of azepine mesoporous carbon of the present invention.
Fig. 2 is the transmission electron microscope picture of azepine mesoporous carbon of the present invention.
Fig. 3 is the assembling of working electrode of the present invention and the hybridization reaction process flow diagram of DNA.
Fig. 4 is horseradish peroxidase enzyme catalytic reaction principle figure.
The curent change curve map that Fig. 5 obtains for detect the Mnp specific coding genetic fragment of variable concentrations with differential pulse voltammetry.
Fig. 6 is the linear regression graph of Mnp specific coding genetic fragment content and curent change.
Embodiment
Embodiment 1
The preparation of the biology sensor based on azepine mesoporous carbon
Biology sensor based on azepine mesoporous carbon of the present invention, for three-electrode electro Chemical sensor, comprise working electrode, contrast electrode and to electrode, described contrast electrode is saturated calomel electrode, described is platinum electrode to electrode, described working electrode comprises glass-carbon electrode, the surface, test side of described glass-carbon electrode is connected with the capture probe of Cys, azepine mesoporous carbon, Au nanoparticles films and sulfydryl modification in turn, is also connected with the mercaptoethanol of sealing Au nanoparticles films residue binding site on described Au nanoparticles films.
The preparation method of described working electrode is,
One, the surface treatment of glass-carbon electrode: by glass-carbon electrode surface finish, then water rinses glass-carbon electrode surface, then carries out ultrasonic cleaning with nitric acid, acetone, water successively, finally rinses with damping fluid again, naturally dries.
Two, the preparation of Cys film: use electrochemical method, add Cys solution, at glass-carbon electrode surface deposition Cys film, the concentration of Cys solution is 1.0 * 10
-3mol/L, obtains Cys modified electrode.
Three, the preparation of modified electrode: by ready azepine mesoporous carbon suspending liquid (with N, dinethylformamide is as spreading agent) be added drop-wise on the surface, test side of Cys modified electrode, naturally dry, then use electrochemical method deposited gold nano particle, obtain modified electrode, naturally dry.
Four, the capture probe of sulfydryl modification is joined on described modified electrode, then add mercaptoethanol, clean, obtain described working electrode, the capture probe of described sulfydryl modification is: 5'-HS-(CH
2)
6-T
1-3', T
1base sequence (being SEQ ID NO.1) be 5'-CTGATGGTGTCGTGTTTCT-3'.
The assemble flow of working electrode as shown in Fig. 3 part, can image the assembly relation of seeing each component.
In azepine mesoporous carbon suspending liquid, azepine mesoporous carbon is to adopt following preparation method to obtain:
(1) synthesising mesoporous silicon template SBA-15: segmented copolymer P123 is placed in to hydrochloric acid and dissolves, then dropwise add ethyl orthosilicate, the mass ratio of segmented copolymer P123 and ethyl orthosilicate is 8: 17, heating water bath after stirring, temperature is controlled at 35 ℃, then gained potpourri is transferred in reactor, at 140 ℃ of heating 25h, suction filtration, washing is to neutral, air-dry, then put into resistance furnace at 550 ℃ of air roasting 5h, obtain mesoporous silicon template SBA-15.
(2) the hybrid mesoporous carbon of synthetic nitrogen: be to add in flask at 0.5: 3: 1.35 according to mass ratio by gained mesoporous silicon template SBA-15, phenixin, ethylenediamine, then at 90～100 ℃, heating water bath stirs 6h, condensing reflux 6h, products therefrom is dried at 60 ℃, be placed in nitrogen thermal treatment 7h at 600 ℃, controlling heating rate is 5 ℃/min again; With the hydrofluorite that massfraction is 7%, remove mesoporous silicon template SBA-15, filter, washing, dry at 40 ℃, obtain azepine mesoporous carbon.
Respectively as shown in Figure 1 and Figure 2, from figure, we can find out for the scanning electron microscope (SEM) photograph of azepine mesoporous carbon of the present invention and transmission electron microscope picture, and azepine mesoporous carbon is strip (SEM), and mesoporous (TEM) in order.
Embodiment 2
The preparation of the biology sensor based on azepine mesoporous carbon
Biology sensor based on azepine mesoporous carbon of the present invention, for three-electrode electro Chemical sensor, comprise working electrode, contrast electrode and to electrode, described contrast electrode is saturated calomel electrode, described is platinum electrode to electrode, described working electrode comprises glass-carbon electrode, the surface, test side of described glass-carbon electrode is connected with the capture probe of Cys, azepine mesoporous carbon, Au nanoparticles films and sulfydryl modification in turn, is also connected with the mercaptoethanol of the residue binding site of sealing Au nanoparticles films on described Au nanoparticles films.
The preparation method of described working electrode is with embodiment 1, and difference is: in second step, the concentration of Cys solution is 1.0 * 10
-2mol/L; In the 3rd step, azepine mesoporous carbon suspending liquid is usingd ultrapure water as spreading agent.
Azepine mesoporous carbon is to adopt the preparation method who comprises the following steps to make:
(1) synthesising mesoporous silicon template SBA-15: segmented copolymer P123 is placed in to hydrochloric acid and dissolves, then dropwise add ethyl orthosilicate, the mass ratio of segmented copolymer P123 and ethyl orthosilicate is 8: 23, heating water bath after stirring, temperature is controlled at 30 ℃, then gained potpourri is transferred in reactor, at 150 ℃ of hydro-thermal 23h, suction filtration, washing is to neutral, air-dry, then put into resistance furnace at 530 ℃ of air roasting 4h, obtain mesoporous silicon template SBA-15;
(2) the hybrid mesoporous carbon of synthetic nitrogen: gained mesoporous silicon template SBA-15, phenixin, ethylenediamine are added in flask according to the mass ratio of 1.5: 3: 1.35, then at 90 ℃～100 ℃, heating water bath stirs 10h, condensing reflux 8h, products therefrom is dried at 40 ℃, be placed in nitrogen and hydrogen thermal treatment 5h at 600 ℃, controlling heating rate is 3 ℃/min again; With the hydrofluorite that massfraction is 5%, remove silicon template, filter, washing, dry at 60 ℃, obtain azepine mesoporous carbon.
Embodiment 3
The method that the described biology sensor based on azepine mesoporous carbon is measured manganese peroxidase specific coding genetic fragment, first the sample drop of the object chain that contains signal probe and variable concentrations is carried out to base complementrity pairing at the working electrode surface of the biology sensor based on azepine mesoporous carbon, reaction is no less than 60 minutes at 37 ℃; Then the phosphate buffered solution that contains nano golden cluster mark horseradish peroxidase-streptavidin (pH is 7.38) is dropped in to the working electrode surface of the biology sensor based on azepine mesoporous carbon, reaction is no less than 30 minutes at 37 ℃; Finally take p-dihydroxy-benzene and hydrogen peroxide as substrate, in being connected to the electrolytic cell of three-electrode system, measure.The curent change value that utilization measures and equation of linear regression calculate the content of manganese peroxidase specific coding genetic fragment; Described equation of linear regression is:
Y=(7.527±0.1787)X+(-66.5603±2.641)
Wherein, Y is current average; X is the natural logarithm of object chain concentration, and unit is molL
-1; Object chain, i.e. manganese peroxidase specific coding genetic fragment, the range of linearity of object chain concentration is 1 * 10
-19～1 * 10
-10m, measurement lower limit is 2 * 10
-20m.
Described signal probe is: 5'-T
2-biotin-3', T
2base sequence (being SEQ ID NO.2) be 5'-GATGCCGTTGTTGGCGGAGAA-3.
The base sequence of object chain (T3) in described liquid to be measured, SEQ ID NO.3 is:
5'-TTCTCCGCCAACAACGGCATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGAAACACGACACCATCAG-3'。
Described nano golden cluster mark horseradish peroxidase-streptavidin adopts the preparation method of following steps to obtain:
(1) streptavidin (4.2mg/mL) of the gold chloride of 0.5mL (100mg/mL) and 1mL horseradish peroxidase-labeled at room temperature mixes while stirring, then when stirring, progressively drip 250 μ L ascorbic acid (4.2mg/mL), stir 45min solution change afterwards muddiness.
(2) 0.1mL NaOH (1M) is added drop-wise in the solution that step (1) obtains, then at room temperature stirs 3 hours, in order to make horseradish peroxidase-streptavidin by nm of gold bunch parcel, solution at room temperature standing 2 hours.
(3) solution step (2) being obtained ultrapure water with 250mL at 4 ℃ is dialysed 4 hours, then uses phosphate buffered solution (pH value is 6.98) dialysis 2 hours, obtains nano golden cluster mark horseradish peroxidase-streptavidin.
The DNA hybridization reaction process flow diagram of capture probe of the present invention, target-probe and signal probe as shown in Figure 3, as can be seen from the figure DNA hybridization reaction flow process relation.
Horseradish peroxidase enzyme catalytic reaction principle as shown in Figure 4, horseradish peroxidase (HRP) is under the condition of p-dihydroxy-benzene and hydrogen peroxide existence, form intermediate product HRP (I) and HRP (II), also has benzoquinone (Q), then benzoquinone is reduced into p-dihydroxy-benzene at electrode surface, produces reduction current.
Embodiment 4
The method that the described biology sensor based on azepine mesoporous carbon is measured manganese peroxidase specific coding genetic fragment, except the preparation method of nano golden cluster mark horseradish peroxidase-streptavidin is with embodiment 3 differences, other steps are identical with embodiment 3.
Described nano golden cluster mark horseradish peroxidase-streptavidin is to adopt the preparation method of following steps to make:
(1) streptavidin (4.2mg/mL) of the gold chloride of 1mL (100mg/mL) and 2mL horseradish peroxidase-labeled at room temperature mixes while stirring, then when stirring again, progressively drip 300 μ L ascorbic acid (4.2mg/mL), stir 30min solution change afterwards muddiness.
(2) 0.4mL NaOH (1M) is added drop-wise in the solution that step (1) obtains, then at room temperature stirs 5 hours, in order to make horseradish peroxidase-streptavidin by nm of gold bunch parcel, solution at room temperature more standing 3 hours.
(3) solution step (2) being obtained ultrapure water with 250mL at 4 ℃ is dialysed 2 hours, and then dialyses 4 hours by phosphate buffered solution (pH is 7.38).
Embodiment 5
Utilize the biology sensor based on azepine mesoporous carbon of the present invention and measure application process 3 groups are measured containing manganese peroxidase (MnP) specific coding genetic fragment testing sample, its measuring process and measurement result are as follows:
1, the preparation of the biology sensor based on azepine mesoporous carbon
With embodiment 1
2, the base complementrity matching method of the described biology sensor based on azepine mesoporous carbon to manganese peroxidase specific coding genetic fragment
The working electrode surface hybridization reaction that three groups of testing samples that contain variable concentrations object chain drop in respectively the biology sensor based on azepine mesoporous carbon is after 60 minutes, by the fluid drips that contains signal probe the working electrode surface hybridization reaction of the biology sensor based on azepine mesoporous carbon 60 minutes, after question response completes, the phosphate buffered solution that contains GNCs-HRP-SA (pH value is 7.38) is dropped in to electrode surface, react 45 minutes at 37 ℃.
3, the demarcation of manganese peroxidase (MnP) specific coding genetic fragment
In the phosphate buffered solution that is 7.38 in pH value, the typical curve of Mnp specific coding genetic fragment is demarcated.Calibration process refers to reacts the working electrode of the biology sensor based on azepine mesoporous carbon successively with the testing sample solution that contains variable concentrations Mnp specific coding genetic fragment, make it carry out base complementrity pairing (course of reaction is identical with above-mentioned steps 2).After having reacted, add certain density p-dihydroxy-benzene and H
2o
2, according to response current, change and draw respectively each group laboratory sample differential pulse voltammetry curve, as shown in Figure 5.And according to the Mnp specific coding genetic fragment concentration of all differential pulse voltammetry curves (DPV curve) and different experiments sample, obtain the natural logarithm relation curve of curent change and Mnp specific coding genetic fragment concentration, as shown in Figure 6, i.e. equation of linear regression, for:
Y=(7.527±0.1787)X+(-66.5603±2.641)
Wherein, Y is current average; X is the natural logarithm of object chain concentration, and unit is molL
-1; The range of linearity of object chain concentration is 1 * 10
-19～1 * 10
-10m, measurement lower limit is 2 * 10
-20m.
4, the mensuration of Mnp specific coding genetic fragment in testing sample
The working electrode surface that three groups of testing samples are added drop-wise to the biology sensor based on azepine mesoporous carbon successively carries out base complementrity match reaction, is then containing 1mmolL
-1in the phosphate buffer of p-dihydroxy-benzene (pH7.38), add 0.5mmolL
-1h
2o
2.With p-dihydroxy-benzene and H
2o
2for substrate, the working electrode of the biology sensor based on azepine mesoporous carbon of take is basal electrode, adopt differential pulse voltammetry, according to the equation of linear regression of setting up in response current variation and step 3, measure Mnp specific coding genetic fragment content in testing sample.It is that the CHI660B electro-chemical systems that adopts Shanghai Chen Hua instrument company to produce is connected with the three-electrode system in 50mL electrolytic cell that described electrochemical process is measured, and controls and monitoring.
Above-mentioned 3 groups of testing samples containing Mnp specific coding genetic fragment are after the biosensor assay of using based on azepine mesoporous carbon, and its measurement result sees the following form:
| Testing sample molL -1 | aMeasure concentration molL -1 | Recovery % |
| 3×10 -11 | 2.89×10 -11 | 96.33 |
| 7×10 -16 | 7.26×10 -16 | 103.71 |
| 9×10 -18 | 8.37×10 -18 | 93 |
Note: a represents the mean concentration of measuring.
Said determination result shows, the present invention is highly sensitive, and selectivity is good, can efficient, low-cost on-line measurement Mnp specific coding genetic fragment content, and for monitoring and the control procedure of microbial degradation lignin provides technical support.
The above is only the preferred embodiment of the present invention, and protection scope of the present invention is also not only confined to above-described embodiment, and all technical schemes belonging under thinking of the present invention all belong to protection scope of the present invention.Be noted that for those skilled in the art, improvements and modifications under the premise without departing from the principles of the invention, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the biology sensor based on azepine mesoporous carbon, comprise working electrode, it is characterized in that, described working electrode comprises glass-carbon electrode, the surface, test side of described glass-carbon electrode is connected with the capture probe of Cys, azepine mesoporous carbon, Au nanoparticles films and sulfydryl modification in turn, is also connected with the mercaptoethanol of sealing Au nanoparticles films residue binding site on described Au nanoparticles films.
2. the biology sensor based on azepine mesoporous carbon as claimed in claim 1, is characterized in that, the capture probe of described sulfydryl modification is: 5'-HS-(CH
2)
6-T
1-3', T
1base sequence be 5'-CTGATGGTGTCGTGTTTCT-3'.
3. the biology sensor based on azepine mesoporous carbon as claimed in claim 1 or 2, it is characterized in that, the preparation method of described azepine mesoporous carbon is, mesoporous silicon material, phenixin, ethylenediamine are mixed, the weight ratio of mesoporous silicon material, phenixin, ethylenediamine is 0.5～1.5:3:1.35, adds thermal agitation, condensing reflux at 90～100 ℃, dry, be placed in nitrogen, or in the mixed gas of nitrogen and hydrogen, be heated to 600～900 ℃, after completion of the reaction, add hydrofluorite, filter, washing, dry, obtain azepine mesoporous carbon.
4. the biology sensor based on azepine mesoporous carbon as claimed in claim 3, it is characterized in that, described mesoporous silicon material is mesoporous silicon template SBA-15, the preparation method of described mesoporous silicon template SBA-15 is, segmented copolymer P123 is placed in to hydrochloric acid to be dissolved, then dropwise add ethyl orthosilicate, the mass ratio of segmented copolymer P123 and ethyl orthosilicate is 8: 17～23, temperature is controlled at 30～35 ℃, stir, obtain potpourri, then by described potpourri 140～150 ℃ of heating, after reacting completely, suction filtration, washing is to neutral, air-dry, again 530～550 ℃ of roastings, obtain mesoporous silicon template SBA-15.
5. the preparation method of the biology sensor based on azepine mesoporous carbon as described in claim 1-4 any one, it is characterized in that, comprise the steps, the preparation method of described working electrode is, on glass-carbon electrode, by electrochemical process, deposit Cys, then add azepine mesoporous carbon suspending liquid, last electrochemical process deposited gold nano particle, obtains modified electrode; The capture probe of sulfydryl modification is joined on described modified electrode, then add mercaptoethanol, clean, obtain described working electrode.
6. the preparation method of the biology sensor based on azepine mesoporous carbon as claimed in claim 5, is characterized in that, the spreading agent of described azepine mesoporous carbon suspending liquid is DMF or ultrapure water.
7. the biology sensor based on azepine mesoporous carbon that as described in the biology sensor based on azepine mesoporous carbon as described in claim 1-4 any one or claim 5-6, preparation method obtains is in the application of measuring manganese peroxidase specific coding genetic fragment concentration, it is characterized in that, step is, the liquid to be measured that contains object chain and the fluid drips that contains signal probe are added in to the working electrode surface of the described biology sensor based on azepine mesoporous carbon, after having reacted, the damping fluid that dropping contains nano golden cluster mark horseradish peroxidase-streptavidin, last in being connected to the electrolytic cell of three-electrode system, take p-dihydroxy-benzene and hydrogen peroxide as substrate, measure, according to equation of linear regression, obtain the concentration of object chain in liquid to be measured, described equation of linear regression is:
Y=(7.527±0.1787)X+(-66.5603±2.641)
Wherein, Y is current average; X is the natural logarithm of object chain concentration, and unit is molL
-1; The range of linearity of object chain concentration is 1 * 10
-19～1 * 10
-10m, measurement lower limit is 2 * 10
-20m.
8. application as claimed in claim 7, is characterized in that, described signal probe is: 5'-T
2-biotin-3', T
2base sequence be 5'-GATGCCGTTGTTGGCGGAGAA-3'; In described liquid to be measured, the base sequence of object chain is: 5'-TTCTCCGCCAACAACGGCATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTAGAAACACGACACCATCAG-3'.
9. application as claimed in claim 7 or 8, it is characterized in that, the preparation method of described nano golden cluster mark horseradish peroxidase-streptavidin is, chlorauric acid solution and horseradish peroxidase-streptavidin solution are mixed, under stirring condition, drip ascorbic acid solution, solution adds strong base solution after becoming muddiness, stir, standing, dialysis, obtain nano golden cluster mark horseradish peroxidase-streptavidin, the quality proportioning of described gold chloride, horseradish peroxidase-streptavidin, ascorbic acid is 50～100:4～9:1～1.5.
As claimed in claim 7 or 8 application, it is characterized in that, described in contain nano golden cluster mark horseradish peroxidase-streptavidin damping fluid be phosphate buffered solution, pH is 6.5～7.5.
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| CN104630869A (en) * | 2015-01-22 | 2015-05-20 | 江南大学 | DNA sensor for detecting staphylococcus aureus as well as preparation method and application of DNA sensor |
| CN104655697A (en) * | 2015-02-04 | 2015-05-27 | 沈阳师范大学 | Preparation method and application of electroactive ionic liquid-based mesoporous silicon modified electrode |
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