CN103743805B - Biosensor based on aza mesoporous carbon, preparation method and applications - Google Patents
Biosensor based on aza mesoporous carbon, preparation method and applications Download PDFInfo
- Publication number
- CN103743805B CN103743805B CN201410022749.6A CN201410022749A CN103743805B CN 103743805 B CN103743805 B CN 103743805B CN 201410022749 A CN201410022749 A CN 201410022749A CN 103743805 B CN103743805 B CN 103743805B
- Authority
- CN
- China
- Prior art keywords
- aza
- electrode
- carbon
- mesoporous carbon
- mesoporous
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The present invention relates to a kind of biosensor based on aza mesoporous carbon, preparation method and applications, including working electrode, described working electrode includes glass-carbon electrode, the surface, test side of described glass-carbon electrode is connected with L cysteine, aza mesoporous carbon, Au nanoparticles films and the capture probe of sulfydryl modification in turn, and described Au nanoparticles films is also associated with mercaptoethanol；The present invention also provides for the application of a kind of biosensor based on aza mesoporous carbon, step is to drip the liquid to be measured containing object chain and the liquid containing signal probe at working electrode surface, the dropping buffer containing nano golden cluster labelling horseradish peroxidase streptavidin, in the electrolyzer being connected to three-electrode system, with hydroquinone and hydrogen peroxide as substrate, measure, the present invention is highly sensitive, response is quickly, accuracy of detection is high, and anti-interference is relatively strong, and application operating is easy, efficiently, testing cost is low.
Description
Technical field
The present invention relates to a kind of biosensor, be specifically related to a kind of bio-sensing based on aza mesoporous carbon
Device, preparation method and applications.
Background technology
In terrestrial ecosystems, lignin is to contain in a kind of amorphous, molecular structure being widely present in plant
There is the armaticity high polymer of oxo phenylpropanol or derivatives thereof construction unit, the most common in agricultural and domestic waste,
And the content in xylophyta accounts for 25%, it is to be only second to the Organic substance that the second of cellulose is the abundantest in the world.Lignin
Natural degradation speed slowly, generally use the method piled up or burn to process, not only can produce such as COx, methane etc.
A large amount of harmful gass, cause the wasting of resources simultaneously, thus Biodegradation of Lignin are more helpful for environmental resource.White rot fungi
Lignin had biodegradability.White rot fungi can secret out of the oxygen such as some such as laccase, manganese peroxidase, LiP
Change exoenzyme lignin degrading, the most especially effect with manganese peroxidase (MnP) the most key.Therefore, the feature of MnP is detected
Encoding gene, the dynamic change of the MnP secreted during can effectively understanding fungal organism lignin degrading, such that it is able to more
Add and control whole biodegradation process accurately and effectively.
Employing biosensor technique detection genetic fragment is trend, wherein an electrochemical sensor of gene analysis test
Paid close attention to widely, because it possesses response quickly, highly sensitive, high selectivity and the advantage such as workable.DNA is passed
Sensor, common working electrode is gold electrode or screen printing electrode based on nanometer gold, because being modified with the gene of sulfydryl
Probe is by the crosslinking of sulfydryl with gold, it is easy to is assemblied in electrode surface, carries out coherent detection.But, gold electrode is expensive,
And screen printing electrode is disposable electrode, for a large amount of detections, it always spends the most considerable.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, it is provided that a kind of response highly sensitive, quick, height
Accuracy of detection and the biosensor based on aza mesoporous carbon compared with common-path interference, the present invention also provides for a kind of simple base
In the preparation method of the biosensor of aza mesoporous carbon, the present invention also provides for a kind of biological biography based on aza mesoporous carbon
Sensor application in terms of manganese peroxidase specific coding genetic fragment measurement of concetration, described application operating is easy, efficiently, surveys
Amount low cost.
The technical scheme that the present invention proposes is,
A kind of biosensor based on aza mesoporous carbon, including working electrode, described working electrode includes glass carbon electricity
Pole, the surface, test side of described glass-carbon electrode is connected with Cys, aza mesoporous carbon, Au nanoparticles films and mercapto in turn
The capture probe that base is modified, described Au nanoparticles films is also associated with close the sulfydryl of Au nanoparticles films residue binding site
Ethanol.
The biosensor based on aza mesoporous carbon of the present invention is preferably three-electrode electrochemical sensor, including work
Electrode, reference electrode and to electrode, described reference electrode is preferably saturated calomel electrode, described electrode is preferably platinum electrode.
Surface, the test side deposition of the described glass-carbon electrode of the present invention has Cys, and Cys connects azepine
Changing mesoporous carbon, connecting outside aza mesoporous carbon has Au nanoparticles films, and described Au nanoparticles films connects sulfydryl modification
Capture probe, described Au nanoparticles films is also associated with close the mercaptoethanol of Au nanoparticles films residue binding site, makes
Golden nanometer particle does not combines with signal probe or manganese peroxidase specific coding genetic fragment etc..
The capture probe of described sulfydryl modification is: 5'-HS-(CH2)6-T1-3', T1Base sequence (i.e. SEQ ID
NO.1) it is 5'-CTGATGGTGTCGTGTTTCT-3'.
The preparation method of described aza mesoporous carbon is, by mesoporous silicon material, carbon tetrachloride, ethylenediamine mixing, mesoporous silicon
Material is preferably mesoporous silicon template SBA-15, and mesoporous silicon material, carbon tetrachloride, the weight ratio of ethylenediamine are preferably 0.5～1.5:
3:1.35, heated and stirred at 90～100 DEG C, the time is preferably 6～10h, condensing reflux, and the time is preferably 6～8h, is preferable over
It is dried under the conditions of 40～60 DEG C, is placed in nitrogen, or nitrogen is with the mixed gas of hydrogen, is heated to 600～900 DEG C of process,
The preferred process time is 5～7h, preferred mode of heating be control heating rate be 3～5 DEG C/min.After being disposed, add
Fluohydric acid., the mass fraction of described Fluohydric acid. is preferably 5～7%, filters, and washing is dried, and baking temperature is preferably 40～60 DEG C,
Obtain aza mesoporous carbon.
Mesoporous silicon template SBA-15 can be prepared by method and obtain, and is placed in hydrochloric acid molten by block copolymer P123
Solving, be then added dropwise over tetraethyl orthosilicate, block copolymer P123 is 8: 17～23 with the mass ratio of tetraethyl orthosilicate, temperature control
System, at 30～35 DEG C, stirs, obtains mixture, then by described mixture after 140～150 DEG C of heating, reaction completely, generally
23～25h, sucking filtration, washing, to neutral, air-dry, then 530～550 DEG C of roastings, obtains mesoporous silicon template SBA-15.
The present invention also provides for the preparation method of a kind of biosensor based on aza mesoporous carbon, described working electrode
Preparation method is, deposits Cys by electrochemical process, be subsequently adding aza mesoporous carbon suspension on glass-carbon electrode,
Last electrochemical process depositing gold nanoparticles, obtains modified electrode；The capture probe of sulfydryl modification is joined described modification electricity
Extremely go up, be subsequently adding mercaptoethanol, clean, obtain described working electrode.
The dispersant of described aza mesoporous carbon suspension is N,N-dimethylformamide or ultra-pure water.
It is specific that the present invention also provides for a kind of described biosensor based on aza mesoporous carbon measurement manganese peroxidase
The application of encoding gene segment concentration, its step is, by the liquid to be measured containing object chain and the dropping of the liquid containing signal probe
At the working electrode surface of described biosensor based on aza mesoporous carbon, after having reacted, dropping is containing nanometer gold group
The buffer of bunch labelling horseradish peroxidase-streptavidin (GNCs-HRP-SA), is finally being connected to the electricity of three-electrode system
Xie Chizhong, with hydroquinone and hydrogen peroxide as substrate, measures, obtains object chain in liquid to be measured according to equation of linear regression
Concentration, 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 logrithm of object chain concentration, and unit is mol L-1；Object chain concentration
The range of linearity is 1 × 10-19～1 × 10-10M, measurement lower limit is 2 × 10-20M。
Described signal probe is: 5'-T2-biotin-3', T2Base sequence (i.e. SEQ ID NO.2) be 5'-
GATGCCGTTGTTGGCGGAGAA-3。
The base sequence (i.e. SEQ ID NO.3) of object chain, i.e. manganese peroxidase specific coding base in described liquid to be measured
Because fragment is:
5'-TTCTCCGCCAACAACGGCATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGAAACACGAC
ACCATCAG-3'。
The preparation method of described nano golden cluster labelling horseradish peroxidase-streptavidin (GNCs-HRP-SA) is,
By chlorauric acid solution and horseradish peroxidase-streptavidin solution mixing, under stirring condition, drip ascorbic acid solution, molten
Liquid adds strong base solution after becoming cloudy, and described strong base solution is preferably sodium hydroxide solution, and stirring stands, dialysis, obtains
GNCs-HRP-SA.Described gold chloride, horseradish peroxidase-streptavidin, the quality proportioning of ascorbic acid be preferably 50～
100:4～9:1～1.5, most preferably 50:4.2:1.05.
The described buffer containing nano golden cluster labelling horseradish peroxidase-streptavidin is that phosphate-buffered is molten
Liquid, pH is preferably 6.5～7.5, most preferably 7.38.
In recent years, along with novel nano-material is rapidly combined with electrochemical sensing technology, in sensor construction strategy, in conjunction with
New material relevant feature, uses some nano materials and biomolecule material to form structure of composite membrane and modifies at glass-carbon electrode
Surface.Nano material, such as golden nanometer particle etc., because having high electronic conductivity, are provided that bigger serface, keep biology
The advantages such as activity, it is considered to be outstanding biomolecule carrier and signal transmission medium, can improve the spirit of electrochemical sensor
Sensitivity.
Compared with prior art, it is an advantage of the current invention that: aza mesoporous carbon has good affine energy to biomolecule
Power, biocompatibility, and there is the conversion of π-π * electronics in aza mesoporous carbon, thus improve electronics pass to ability.L-
Cysteine is nontoxic, biocompatibility is strong, have film forming ability.
Nano golden cluster labelling horseradish peroxidase-streptavidin amplifies material as signal, because nanometer gold group
Bunch core itself has catalysis and the ability of conduction electronics, has equally at the horseradish peroxidase-streptavidin wrapped up about
Catalytic action, therefore, nano golden cluster labelling horseradish peroxidase-streptavidin has dual signal expansion effect, enters
And improve the sensitivity of sensor.
The present invention has taken into full account aza mesoporous carbon, nanometer gold, Cys, nano golden cluster labelling Radix Cochleariae officinalis peroxide
Compound enzyme-respective character of streptavidin, and utilize the composite membrane that they form, build response highly sensitive, quick, high detection
Precision and the biosensor based on aza mesoporous carbon compared with common-path interference, described biological biography based on aza mesoporous carbon
Sensor is used for detecting MnP genetic fragment, and easy and simple to handle, efficiently, testing cost is low, for monitoring and the control of microbial degradation lignin
Process processed provides a kind of effective biology tool.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope (SEM) photograph of the aza mesoporous carbon of the present invention.
Fig. 2 is the transmission electron microscope picture of the aza mesoporous carbon of the present invention.
Fig. 3 is the assembling hybridization flow chart with DNA of the working electrode of the present invention.
Fig. 4 is horseradish peroxidase enzyme catalytic reaction principle figure.
Fig. 5 is the curent change obtained by the Mnp specific coding genetic fragment of differential pulse voltammetry detection variable concentrations
Curve chart.
Fig. 6 is the linear regression graph of Mnp specific coding genetic fragment content and curent change.
Detailed description of the invention
Embodiment 1
The preparation of biosensor based on aza mesoporous carbon
The biosensor based on aza mesoporous carbon of the present invention, for three-electrode electrochemical sensor, including work electricity
Pole, reference electrode and to electrode, described reference electrode is saturated calomel electrode, and described is platinum electrode to electrode, described work electricity
Pole includes glass-carbon electrode, and the surface, test side of described glass-carbon electrode is connected with Cys, aza mesoporous carbon, Jenner in turn
Rice corpuscles film and the capture probe of sulfydryl modification, described Au nanoparticles films is also associated with close Au nanoparticles films residue knot
Close the mercaptoethanol in site.
The preparation method of described working electrode is,
One, the surface of glass-carbon electrode processes: glassy carbon electrode surface is polished, then rinses glassy carbon electrode surface with water, then
Carry out ultrasonic cleaning with nitric acid, acetone, water successively, the most again with wash buffer, naturally dry.
Two, the preparation of Cys film: by the method for electrochemistry, add Cys solution, at glass-carbon electrode table
Face deposition Cys film, the concentration of Cys solution is 1.0 × 10-3Mol/L, obtains Cys and modifies electricity
Pole.
Three, the preparation of modified electrode: by ready aza mesoporous carbon suspension (using N,N-dimethylformamide as
Dispersant) it is added drop-wise on the surface, test side of Cys modified electrode, naturally dry, then with the method deposition gold of electrochemistry
Nanoparticle, obtains modified electrode, naturally dries.
Four, the capture probe of sulfydryl modification is joined on described modified electrode, be subsequently adding mercaptoethanol, clean,
To described working electrode, the capture probe of described sulfydryl modification is: 5'-HS-(CH2)6-T1-3', T1Base sequence (i.e. SEQ
ID NO.1) it is 5'-CTGATGGTGTCGTGTTTCT-3'.
The assemble flow of working electrode as partially shown in figure 3, can image the assembly relation seeing each component.
In aza mesoporous carbon suspension, aza mesoporous carbon is to use following preparation method to obtain:
(1) synthesising mesoporous silicon template SBA-15: block copolymer P123 is placed in hydrochloric acid dissolving, is just then being added dropwise over
Silester, block copolymer P123 is 8: 17 with the mass ratio of tetraethyl orthosilicate, and heating in water bath after stirring, temperature controls 35
DEG C, then gained mixture being transferred in reactor, heat 25h, sucking filtration at 140 DEG C, washing, to neutral, air-dry, places into
In resistance furnace in 550 DEG C of air roasting 5h, obtain mesoporous silicon template SBA-15.
(2) the hybrid mesoporous carbon of synthetic nitrogen: by gained mesoporous silicon template SBA-15, carbon tetrachloride, ethylenediamine according to mass ratio
Being in 0.5: 3: 1.35 addition flask, then heating in water bath stirring 6h at 90～100 DEG C, condensing reflux 6h, by products therefrom
Being dried at 60 DEG C, then be placed in nitrogen heat treatment 7h at 600 DEG C, controlling heating rate is 5 DEG C/min；Divide by quality
Number is Fluohydric acid. removing mesoporous silicon template SBA-15 of 7%, filters, washing, is dried, obtains aza mesoporous carbon at 40 DEG C.
The scanning electron microscope (SEM) photograph of the aza mesoporous carbon of the present invention and transmission electron microscope picture are distinguished the most as shown in Figure 1 and Figure 2, from figure,
It will be seen that aza mesoporous carbon is strip (SEM), and mesoporous orderly (TEM).
Embodiment 2
The preparation of biosensor based on aza mesoporous carbon
The biosensor based on aza mesoporous carbon of the present invention, for three-electrode electrochemical sensor, including work electricity
Pole, reference electrode and to electrode, described reference electrode is saturated calomel electrode, and described is platinum electrode to electrode, described work electricity
Pole includes glass-carbon electrode, and the surface, test side of described glass-carbon electrode is connected with Cys, aza mesoporous carbon, Jenner in turn
Rice corpuscles film and the capture probe of sulfydryl modification, described Au nanoparticles films is also associated with close the residue of Au nanoparticles films
The mercaptoethanol of binding site.
The preparation method of described working electrode is with embodiment 1, and difference is: in second step, Cys solution dense
Degree is 1.0 × 10-2mol/L；In 3rd step, aza mesoporous carbon suspension is using ultra-pure water as dispersant.
Aza mesoporous carbon is to use the preparation method comprised the following steps to prepare:
(1) synthesising mesoporous silicon template SBA-15: block copolymer P123 is placed in hydrochloric acid dissolving, is just then being added dropwise over
Silester, block copolymer P123 is 8: 23 with the mass ratio of tetraethyl orthosilicate, and heating in water bath after stirring, temperature controls 30
DEG C, then gained mixture is transferred in reactor, at 150 DEG C of hydro-thermals 23h, sucking filtration, washing, to neutral, air-dry, places into
In resistance furnace in 530 DEG C of air roasting 4h, obtain mesoporous silicon template SBA-15；
(2) the hybrid mesoporous carbon of synthetic nitrogen: by gained mesoporous silicon template SBA-15, carbon tetrachloride, ethylenediamine according to 1.5: 3:
The mass ratio of 1.35 adds in flask, and then heating in water bath stirring 10h, condensing reflux 8h at 90 DEG C～100 DEG C, produces gained
Thing is dried at 40 DEG C, then is placed in nitrogen and hydrogen heat treatment 5h at 600 DEG C, and controlling heating rate is 3 DEG C/min；
By the Fluohydric acid. removing silicon template that mass fraction is 5%, filter, washing, be dried at 60 DEG C, obtain aza mesoporous carbon.
Embodiment 3
Manganese peroxidase specific coding genetic fragment is surveyed by described biosensor based on aza mesoporous carbon
The method of amount, first by the sample drop of the object chain containing signal probe and variable concentrations at biology based on aza mesoporous carbon
The working electrode surface of sensor carries out base pair complementarity, and at 37 DEG C, reaction was no less than 60 minutes；Then will be containing nanometer gold
The phosphate buffered solution (pH is 7.38) of cluster labelling horseradish peroxidase-streptavidin drops in based on aza mesoporous
The working electrode surface of the biosensor of carbon, at 37 DEG C, reaction was no less than 30 minutes；Last with hydroquinone and hydrogen peroxide
For substrate, measure in the electrolyzer be connected to three-electrode system.Utilize and measure the current variation value and linear regression side arrived
Journey calculates 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 logrithm of object chain concentration, and unit is mol L-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'-T2-biotin-3', T2Base sequence (i.e. SEQ ID NO.2) be 5'-
GATGCCGTTGTTGGCGGAGAA-3。
The base sequence of object chain (T3) in described liquid to be measured, i.e. SEQ ID NO.3 is:
5'-TTCTCCGCCAACAACGGCATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGAAACACGAC
ACCATCAG-3'。
Described nano golden cluster labelling horseradish peroxidase-streptavidin uses the preparation method of following steps to obtain
Arrive:
(1) gold chloride (100mg/mL) of 0.5mL and the streptavidin (4.2mg/ of 1mL horseradish peroxidase-labeled
ML) mix the most while stirring, then while stirring, progressively drip 250 μ L ascorbic acid (4.2mg/mL), stirring
Solution turned cloudy after 45min.
(2) 0.1mL sodium hydroxide (1M) is added drop-wise in the solution that step (1) obtains, is then stirred at room temperature 3 little
Time, in order to make horseradish peroxidase-streptavidin be wrapped up by nanometer gold bunch, solution at room temperature stands 2 hours.
(3) solution step (2) obtained is dialysed 4 hours with the ultra-pure water of 250mL at 4 DEG C, then delays with phosphate
Dissolved liquid (pH value is 6.98) is dialysed 2 hours, obtains nano golden cluster labelling horseradish peroxidase-streptavidin.
The DNA hybridization reacting flow chart of the capture probe of the present invention, target-probe and signal probe is as it is shown on figure 3, from figure
In can be seen that DNA hybridization reaction process relation.
As shown in Figure 4, horseradish peroxidase (HRP) is in hydroquinone and mistake for horseradish peroxidase enzyme catalytic reaction principle
Under conditions of hydrogen oxide exists, forming intermediate product HRP (I) and HRP (II), also benzoquinone (Q), then benzoquinone exists
Electrode surface is reduced into hydroquinone, produces reduction current.
Embodiment 4
Manganese peroxidase specific coding genetic fragment is surveyed by described biosensor based on aza mesoporous carbon
The method of amount, except the preparation method of nano golden cluster labelling horseradish peroxidase-streptavidin is different with embodiment 3
Outward, other steps are identical with embodiment 3.
Described nano golden cluster labelling horseradish peroxidase-streptavidin is the preparation method system using following steps
:
(1) gold chloride (100mg/mL) of 1mL and the streptavidin (4.2mg/mL) of 2mL horseradish peroxidase-labeled
Mix the most while stirring, while being stirred for, then progressively drip 300 μ L ascorbic acid (4.2mg/mL), stirring
Solution turned cloudy after 30min.
(2) 0.4mL sodium hydroxide (1M) is added drop-wise in the solution that step (1) obtains, is then stirred at room temperature 5 little
Time, in order to make horseradish peroxidase-streptavidin be wrapped up by nanometer gold bunch, solution stands 3 hours the most again.
(3) solution step (2) obtained is dialysed 2 hours with the ultra-pure water of 250mL at 4 DEG C, uses phosphate the most again
Buffer solution (pH is 7.38) is dialysed 4 hours.
Embodiment 5
Utilize the biosensor based on aza mesoporous carbon of the present invention and measure application process to 3 groups of peroxidating in manganese
Thing enzyme (MnP) specific coding genetic fragment testing sample is measured, and its measuring process and measurement result are as follows:
1, the preparation of biosensor based on aza mesoporous carbon
With embodiment 1
2, the described biosensor based on the aza mesoporous carbon alkali to manganese peroxidase specific coding genetic fragment
Base complementary pairing method
Three groups of testing samples containing variable concentrations object chain drop in biosensor based on aza mesoporous carbon respectively
Working electrode surface hybridization after 60 minutes, the liquid containing signal probe is dropped in biology based on aza mesoporous carbon
The working electrode surface hybridization of sensor 60 minutes, after question response completes, by the phosphate-buffered containing GNCs-HRP-SA
Solution (pH value is 7.38) drops in electrode surface, reacts 45 minutes at 37 DEG C.
3, the demarcation of manganese peroxidase (MnP) specific coding genetic fragment
In the phosphate buffered solution that pH value is 7.38, the standard curve of Mnp specific coding genetic fragment is marked
Fixed.Calibration process refer to by the working electrode of biosensor based on aza mesoporous carbon successively with containing variable concentrations Mnp
The testing sample solution reaction of specific coding genetic fragment so that it is carry out base pair complementarity (course of reaction and above-mentioned steps 2 phase
With).Certain density hydroquinone and H is added after having reacted2O2, draw each group of experiment respectively according to response current change
Sample differential pulse voltammetry curve, as shown in Figure 5.And it is real according to all of differential pulse voltammetry curve (DPV curve) and difference
Test the Mnp specific coding genetic fragment concentration of sample, obtain the most right of curent change and Mnp specific coding genetic fragment concentration
Number relation curve, 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 logrithm of object chain concentration, and unit is mol L-1；Object chain concentration
The range of linearity 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 biosensor based on aza mesoporous carbon successively is carried out
Base pair complementarity reacts, then containing 1mmol L-1The phosphate buffer (pH7.38) of hydroquinone adds
0.5mmol·L-1H2O2.With hydroquinone and H2O2For substrate, the work with biosensor based on aza mesoporous carbon is electric
Extremely basal electrode, uses differential pulse voltammetry, according to the equation of linear regression set up in response current change and step 3,
Measure Mnp specific coding genetic fragment content in testing sample.It is to use Shanghai occasion China instrument public that described electrochemical process measures
The CHI660B electro-chemical systems that department produces is connected with the three-electrode system in 50mL electrolyzer, is controlled and monitors.
Above-mentioned 3 groups of testing samples containing Mnp specific coding genetic fragment are with bio-sensing based on aza mesoporous carbon
After device measures, its measurement result see table:
| Testing sample mol L-1 | aMeasure concentration mol L-1 | Response rate % |
| 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 measurement.
Said determination result shows, the present invention is highly sensitive, and selectivity is good, it is possible to efficient, low cost on-line measurement Mnp spy
Determining encoding gene segment content, the monitoring and control process for 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 not limited merely to above-mentioned enforcement
Example, all technical schemes belonged under thinking of the present invention belong to protection scope of the present invention.It it is noted that for the art
Those of ordinary skill for, improvements and modifications under the premise without departing from the principles of the invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (6)
1. manganese peroxidase specific coding genetic fragment concentration measured by a biosensor based on aza mesoporous carbon
Application, it is characterized in that, described biosensor based on aza mesoporous carbon includes that working electrode, described working electrode include
Glass-carbon electrode, the surface, test side of described glass-carbon electrode is connected with Cys, aza mesoporous carbon, golden nanometer particle in turn
Film and the capture probe of sulfydryl modification, described Au nanoparticles films is also associated with close Au nanoparticles films residue binding site
Mercaptoethanol；The capture probe of described sulfydryl modification is: 5'-HS-(CH2)6-T1-3', T1Base sequence be 5'-
CTGATGGTGTCGTGTTTCT-3'；
Described application process is: drip described the liquid to be measured containing object chain and the liquid containing signal probe based on azepine
Changing the working electrode surface of the biosensor of mesoporous carbon, after having reacted, dropping is containing nano golden cluster labelling Radix Cochleariae officinalis peroxide
The buffer of compound enzyme-streptavidin, finally in the electrolyzer being connected to three-electrode system, with hydroquinone and hydrogen peroxide
For substrate, measuring, obtain the concentration of object chain in liquid to be measured according to equation of linear regression, described equation of linear regression is:
；
Wherein, Y is current average；X is the natural logrithm of object chain concentration, and unit is mol L-1；Object chain concentration linear
Scope is 1 × 10−19～1 × 10−10 M, measurement lower limit is 2 × 10−20 M；
Described signal probe is: 5'-T2-biotin-3', T2Base sequence be 5'-GATGCCGTTGTTGGCGGAGAA-3'；
In described liquid to be measured, the base sequence of object chain is: 5'-TTCTCCGCCAACAACGGCATCTTTTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTTTAGAAACACGACACCATCAG-3'；
The preparation method of described nano golden cluster labelling horseradish peroxidase-streptavidin is, by chlorauric acid solution and peppery
Root peroxidase-streptavidin solution mixing, drips ascorbic acid solution under stirring condition, add strong after solution turned cloudy
Aqueous slkali, stirring, stand, dialysis, obtain nano golden cluster labelling horseradish peroxidase-streptavidin, described gold chloride,
Horseradish peroxidase-streptavidin, the quality proportioning of ascorbic acid are 50～100:4～9:1～1.5.
Applying the most as claimed in claim 1, it is characterized in that, the preparation method of described aza mesoporous carbon is, by mesoporous silicon material
Material, carbon tetrachloride, ethylenediamine mix, and mesoporous silicon material, carbon tetrachloride, the weight ratio of ethylenediamine are 0.5～1.5:3:1.35,
Heated and stirred at 90～100 DEG C, condensing reflux, it is dried, is placed in nitrogen, or nitrogen is with the mixed gas of hydrogen, is heated to
600～900 DEG C, after completion of the reaction, add Fluohydric acid., filter, washing, it is dried, obtains aza mesoporous carbon.
Applying the most as claimed in claim 2, it is characterized in that, described mesoporous silicon material is mesoporous silicon template SBA-15, is given an account of
The preparation method of hole silicon template SBA-15 is, block copolymer P123 is placed in hydrochloric acid dissolving, is then added dropwise over positive silicic acid
Ethyl ester, block copolymer P123 is 8:17～23 with the mass ratio of tetraethyl orthosilicate, and temperature controls at 30～35 DEG C, stirring,
Mixture, then by described mixture after 140～150 DEG C of heating, reaction completely, sucking filtration, washing, to neutral, air-dries, then
530～550 DEG C of roastings, obtain mesoporous silicon template SBA-15.
4. having the application described in 1 according to claim, it is characterized in that, the preparation method of described working electrode is, at glass-carbon electrode
On deposit Cys by electrochemical process, be subsequently adding aza mesoporous carbon suspension, last electrochemical process deposition Jenner
Rice corpuscles, obtains modified electrode；The capture probe of sulfydryl modification is joined on described modified electrode, is subsequently adding sulfydryl second
Alcohol, cleans, obtains described working electrode.
Application the most according to claim 4, is characterized in that, the dispersant of described aza mesoporous carbon suspension is N, N-bis-
Methylformamide or ultra-pure water.
Apply the most as claimed in claim 1, it is characterized in that, described containing nano golden cluster labelling horseradish peroxidase-chain
The buffer of enzyme Avidin is phosphate buffered solution, and pH is 6.5～7.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410022749.6A CN103743805B (en) | 2014-01-17 | 2014-01-17 | Biosensor based on aza mesoporous carbon, preparation method and applications |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410022749.6A CN103743805B (en) | 2014-01-17 | 2014-01-17 | Biosensor based on aza mesoporous carbon, preparation method and applications |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103743805A CN103743805A (en) | 2014-04-23 |
| CN103743805B true CN103743805B (en) | 2016-07-27 |
Family
ID=50500843
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410022749.6A Expired - Fee Related CN103743805B (en) | 2014-01-17 | 2014-01-17 | Biosensor based on aza mesoporous carbon, preparation method and applications |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103743805B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104165915B (en) * | 2014-08-18 | 2016-05-04 | 湖南大学 | A kind of for detection of silver-colored biology sensor and its preparation method and application |
| CN104483366B (en) * | 2014-12-05 | 2017-02-22 | 湖南大学 | Biosensor for detecting lead, as well as preparation method and application of biosensor for detecting lead |
| CN104630869B (en) * | 2015-01-22 | 2017-07-14 | 江南大学 | A kind of DNA sensor for detecting staphylococcus aureus and its preparation and application |
| CN104655697B (en) * | 2015-02-04 | 2017-05-31 | 沈阳师范大学 | A kind of preparation method and application of electroactive ionic liquid base mesoporous silicon modified electrode |
| CN105092683B (en) * | 2015-08-31 | 2018-02-23 | 湖南大学 | It is a kind of to be used to detect electrochemical sensor of lead and its preparation method and application |
| CN107085025B (en) * | 2016-02-15 | 2019-09-17 | 太原师范学院 | A kind of self assembly trilamellar membrane electrode and its preparation method and application |
| CN109312384B (en) | 2016-06-15 | 2022-12-30 | 伊士曼化工公司 | Physical vapor deposition biosensor assembly |
| KR102547063B1 (en) | 2016-09-16 | 2023-06-22 | 이스트만 케미칼 컴파니 | Biosensor electrodes fabricated by physical vapor deposition |
| EP3512958B1 (en) | 2016-09-16 | 2022-04-06 | Eastman Chemical Company | Biosensor electrodes prepared by physical vapor deposition |
| US11881549B2 (en) | 2017-06-22 | 2024-01-23 | Eastman Chemical Company | Physical vapor deposited electrode for electrochemical sensors |
| CN108455557B (en) * | 2018-03-09 | 2019-11-08 | 中南林业科技大学 | Nitrogen sulphur codope ordered mesoporous carbon material and its preparation method and application |
| CN109490385B (en) * | 2018-08-08 | 2020-12-11 | 上海市浦东医院 | Biosensor based on Au-ZIF-8/OMC mesoporous carbon |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102253092A (en) * | 2011-04-19 | 2011-11-23 | 湖南大学 | Composite film modified DNA sensor and its preparation method and application in detection of lignin peroxidase (Lip) specific coding gene segment |
| CN102854233A (en) * | 2012-09-17 | 2013-01-02 | 湖南大学 | Modified-electrode-based tyrosinase biosensor as well as preparation method and application thereof |
| CN103389328A (en) * | 2013-07-24 | 2013-11-13 | 湖南大学 | Tyrosinase biosensor based on modified electrode, and preparation method and application thereof |
-
2014
- 2014-01-17 CN CN201410022749.6A patent/CN103743805B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102253092A (en) * | 2011-04-19 | 2011-11-23 | 湖南大学 | Composite film modified DNA sensor and its preparation method and application in detection of lignin peroxidase (Lip) specific coding gene segment |
| CN102854233A (en) * | 2012-09-17 | 2013-01-02 | 湖南大学 | Modified-electrode-based tyrosinase biosensor as well as preparation method and application thereof |
| CN103389328A (en) * | 2013-07-24 | 2013-11-13 | 湖南大学 | Tyrosinase biosensor based on modified electrode, and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| A sandwich-type DNA biosensor based on electrochemical co-reduction synthesis of graphene-three dimensional nanostructure gold nanocomposite films;Ai-Lin Liu等;《Analytica Chimica Acta》;20130108;第767卷;第52页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103743805A (en) | 2014-04-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103743805B (en) | Biosensor based on aza mesoporous carbon, preparation method and applications | |
| Roushani et al. | Electroanalytical sensing of Cd2+ based on metal–organic framework modified carbon paste electrode | |
| CN102910621B (en) | Preparation methods and applications of multilayer graphene and multilayer graphene modified electrode | |
| Khan et al. | Electrochemical determination of uric acid in the presence of ascorbic acid on electrochemically reduced graphene oxide modified electrode | |
| CN108169303B (en) | Aptamer electrochemical sensor based on metal-organic framework material as signal probe | |
| Du et al. | Ionic liquid-functionalized graphene as modifier for electrochemical and electrocatalytic improvement: comparison of different carbon electrodes | |
| CN101982764B (en) | Composite membrane modified biosensor and preparation method and application thereof | |
| CN109490385B (en) | Biosensor based on Au-ZIF-8/OMC mesoporous carbon | |
| CN104198559B (en) | Electrochemica biological sensor and preparation method for detection of organic phosphorus pesticide | |
| CN103389328B (en) | Based on tyrosinase biology sensor and the preparation method and application of modified electrode | |
| CN104483366A (en) | Biosensor for detecting lead, as well as preparation method and application of biosensor for detecting lead | |
| Fu et al. | Electrochemiluminescence sensor for dopamine with a dual molecular recognition strategy based on graphite-like carbon nitride nanosheets/3, 4, 9, 10-perylenetetracarboxylic acid hybrids | |
| CN104020204A (en) | Electrochemical sensor for detecting lead as well as preparation method and application thereof | |
| CN107238645A (en) | On-line monitoring glucose oxidase screen printing electrode and preparation method thereof | |
| CN103616418A (en) | DNA (Deoxyribonucleic Acid) electrochemical biosensor and preparation method thereof | |
| CN105954346A (en) | Preparation method of a three-dimensional paper-based electrochemical ratio meter | |
| CN105092683A (en) | Electrochemical sensor for detecting lead and preparation method and application of electrochemical sensor | |
| Lin et al. | Monolayer modification of glassy carbon electrode by using propionylcholine for selective detection of uric acid | |
| CN105445346A (en) | Construction method of photoelectrochemical adaptor sensor based on gold/zinc oxide composite and bisphenol A detection method | |
| Shi et al. | Enhanced solid-state electrogenerated chemiluminescence of Au/CdS nanocomposite and its sensing to H2O2 | |
| CN110308188A (en) | Golden co-modified Graphene electrodes of platinum and its preparation method and application | |
| Stilman et al. | Detection of yeast strains by combining surface-imprinted polymers with impedance-based readout | |
| CN106442649A (en) | Method for detecting 1, 5-dehydrated glucose alcohol based on EIS structure electrochemical biosensor | |
| CN109085222A (en) | Ion liquid functionalization graphene vibrios DNA electrochemical sensor and its preparation method and application | |
| Dong et al. | Flexible carbon fiber cloth supports decorated with cerium metal-organic frameworks and multi-walled carbon nanotubes for simultaneous on-site detection of Cd2+ and Pb2+ in food and water samples |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160727 Termination date: 20190117 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |