CN102253092A - Composite film modified DNA sensor and its preparation method and application in detection of lignin peroxidase (Lip) specific coding gene segment - Google Patents
Composite film modified DNA sensor and its preparation method and application in detection of lignin peroxidase (Lip) specific coding gene segment Download PDFInfo
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Abstract
The invention discloses a composite film modified DNA sensor. The DNA sensor is characterized in that the sensor comprises a carbon paste electrode; the carbon paste electrode comprises a carbon rod, a Teflon tube, a magnet, and carbon paste; an induction end of the carbon paste electrode is coated with a sensitive substance; the sensitive substance comprises a composite film and a DNA capture probe; magnetic nanoparticles, multi-walled carbon nanoscale tube-gold nanoparticles and chitosan are utilized for modifying orderly the surface of the carbon paste electrode and compose the composite film; and the DNA capture probe is utilized for modifying the surface of the multi-walled carbon nanoscale tube-gold nanoparticles. The invention also discloses a preparation method of the DNA sensor. The preparation method comprises the processing steps of manufacturing a carbon paste electrode, modifying the surface of an induction end of the manufactured carbon paste electrode by a sensitive substance, and the like. The invention further discloses an application of the DNA sensor in detecting a lignin peroxidase (Lip) specific coding gene segment. Because a sensitive substance is utilized for modifying the surface of a carbon paste electrode of the DNA sensor, the DNA sensor has a strong capacity of electron conduction and a high accuracy of detection. The preparation method has the advantages of low cost and simple process.
Description
Technical field
The present invention relates to field of biosensors, relate in particular to a kind of process biology sensor after film modified and its production and application.
Background technology
Adopting biosensor technique to detect genetic fragment is a trend of gene analysis test, and wherein electrochemical sensor is subjected to paying close attention to widely, highly sensitive because it possesses response fast, high selectivity and advantage such as workable.For the DNA sensor, common working electrode is a gold electrode or based on the screen printing electrode of nm of gold, because the gene probe that is modified with sulfydryl crosslinked by sulfydryl and gold is easy to be assemblied in electrode surface, carries out coherent detection.But, gold electrode costs an arm and a leg, and the screen printing electrode is a disposable electrode, and for a large amount of detections, its total cost is also very considerable.In recent years, along with novel nano-material combines with the electrochemical sensing technology is swift and violent, a class is developed rapidly based on the electrochemical sensor of paramagnetism technique for fixing.This class sensor is filled into carbon paste in the polytetrafluoroethylene (PTFE) pipe, and in the carbon paste of magnet intercalation electrode front portion, be formed on the carbon paste electrode that the electrode front end surface has fixed magnetic field, and then, utilize paramagnetism to attract the magnetic nanoparticle of fixed functionization again, make up the specific detection that different sensors is used for the related objective thing.From the operating cost angle, adopt economic magnetic nanoparticle and carbon paste electrode to replace above-mentioned two class electrodes to carry out genetic fragment and detect a kind of feasible way of can yet be regarded as.But have a problem can not be ignored: the electronic conduction ability of carbon paste electrode is starkly lower than gold electrode, can reduce the accuracy of detection of gene sensing technology.
In order to improve the electronic conduction ability of carbon paste electrode, in the sensor construction strategy, learn relevant feature in conjunction with new material, use some nano materials and biomolecule material to form structure of composite membrane and be modified at the carbon paste electrode surface.Nano material, for example carbon nano-tube, magnetic nano-particle and golden nanometer particle etc. can provide bigger serface because have high electronic conductivity, keep advantages such as biologically active, be considered to outstanding biomolecule carrier and signal and transmit media, can improve the sensitivity of electrochemical sensor.Except nano material, a kind of biomolecule material, shitosan is strong because of nontoxic, biological fitness, have characteristics such as film forming ability also is widely used in the structure of biology sensor.
Summary of the invention
Technical matters to be solved by this invention is: at the deficiencies in the prior art, provide the complex film modified DNA sensor that a kind of cost is low, electronic conduction ability is strong, accuracy of detection is high, also corresponding preparation method and the application of this DNA sensor in detecting lignin peroxidase (Lip) specific coding genetic fragment that a kind of this DNA sensor is provided.
For solving the problems of the technologies described above, the present invention by the following technical solutions:
A kind of complex film modified DNA sensor, comprise a carbon paste electrode, described carbon paste electrode comprises carbon-point and PTFE tube, described carbon-point is built in the described PTFE tube, described carbon-point one end is drawn described PTFE tube by electric wire, the described carbon-point other end contacts with a magnet, be filled with carbon paste between the described magnet and the described PTFE tube mouth of pipe, the induction end of described carbon paste electrode is coated with sensitive materials, described sensitive materials comprise the DNA capture probe of composite membrane and sulfhydrylation, described composite membrane is by modifying successively in the magnetic nanoparticle of the sulfhydrylation on described carbon paste electrode surface, multi-walled carbon nano-tubes-the golden nanometer particle of sulfhydrylation, and the shitosan composition, described DNA capture probe is modified in described multi-walled carbon nano-tubes-golden nanometer particle surface.
As further improvement of these options, described magnet is arranged at apart from the mouth of pipe of described polyfluortetraethylene pipe and is not more than the 8mm place.
In the technique scheme, the sequence preference of described DNA capture probe is 5 '-HS (CH
2)
6TTGTTGACGAAGGACTGCCA-3 '.
As a total technical conceive, the present invention also provides a kind of preparation method of above-mentioned complex film modified DNA sensor, may further comprise the steps:
(1) makes carbon paste electrode: in PTFE tube, put into carbon-point, and insert magnet at described carbon-point one end, form magnetic regions, fill with carbon paste between the described magnet and the PTFE tube mouth of pipe, the carbon-point other end is drawn PTFE tube by electric wire, obtain the crude green body of carbon paste electrode, more described crude green body is carried out surface treatment, obtain final carbon paste electrode;
(2) decorating carbon paste electrode surface: the induction end surface of the magnetic nanoparticle for preparing and multi-walled carbon nano-tubes-golden nanometer particle and chitosan solution being modified successively the carbon paste electrode that step (1) makes, naturally obtain complex film modified carbon paste electrode after drying, DNA capture probe with ready sulfhydrylation drops in through complex film modified carbon paste electrode induction end surface again, after the self assembly, obtain complex film modified DNA sensor.
The technological process of above-mentioned preparation magnetic nanoparticle is preferably: prepare Fe under nitrogen protection
3O
4Gelatinous precipitate adds polyglycol, positive silane ethyl ester, methyl alcohol, aminopropyl trimethoxysilane and halfcystine then successively, obtains the magnetic nanoparticle of sulfhydrylation after the reaction.
The technological process of above-mentioned preparation multi-walled carbon nano-tubes-golden nanometer particle is preferably: with the multi-walled carbon nano-tubes purifying, react with halfcystine again, make the multi-walled carbon nano-tubes sulfhydrylation, with the multi-walled carbon nano-tubes and the golden nanometer particle reaction of sulfhydrylation, obtain the multi-walled carbon nano-tubes-golden nanometer particle of sulfhydrylation then.
The self assembly principle of the modification of the composite membrane of DNA sensor of the present invention and DNA capture probe as shown in Figure 2.
As a total inventive concept, the present invention also provide utilize above-mentioned complex film modified DNA sensor (sequence of DNA capture probe is 5 '-HS (CH
2)
6TTGTTGACGAAGGACTGCCA-3 ') method that Lip specific coding genetic fragment is detected may further comprise the steps:
(1) choose the sequence of object chain, and the sequence of modelled signal probe:
The sequence of object chain is 5 '-TGGCAGTCCTTCGTCAACAA-3 '; According to the sequence of object chain, the sequence of modelled signal probe is 5 '-Biotin-TGGCAGTCCTTCGTCAACAA-3 ';
(2) competitive hybridization reaction: described signal probe and solution to be measured are splashed into the induction end surface of described complex film modified DNA sensor, and competitive reaction is no less than 60 minutes;
(3) signalase amplifies: again pH is DNA sensor surface neutral, that phosphate buffered solution that contain Avidin-horseradish peroxidase (SA-HRP) drops in the competitive hybridization reaction, physiological temp (for example 37 ℃) reaction down is no less than 30 minutes, forms signalase connection amplification system;
(4) enzymic catalytic reaction: at last with benzenediol and H
2O
2Be substrate, with the DNA sensor that contains described horseradish peroxidase (HRP) signalase connection amplification system is working electrode, set up the electrolytic cell of three-electrode system, adopt the reduction current that produces in the chronoamperometry mensuration enzyme-catalyzed reaction to change, if the reduction current fall is not less than 6.9%, judge and contain lignin peroxidase specific coding genetic fragment in the solution to be measured, finish detection.
Above-mentioned detection method is based on following principle (as shown in Figure 3) and finishes: the HRP of mark on the signal probe, and can be at H
2O
2Catalytic decomposition p-dihydroxy-benzene under the condition that exists is when add p-dihydroxy-benzene and H in electrolytic cell
2O
2After, HRP makes a hydroxyl on the p-dihydroxy-benzene lose H
+Become the carbonyl of two keys, provide electronics to make its reduction by electrode surface again, this process can produce detectable response signal and reach identifying purpose, and according to the content of the strong and weak corresponding Lip specific coding genetic fragment of the signal that is produced, thereby realize mensuration to Lip specific coding genetic fragment.
In the technique scheme, the reduction potential that detects in the described electrolytic cell is preferably-0.3V～-0.2V, most preferably be-0.252V, described electrolytic solution cell is preferably the neutral phosphate buffered solution of pH value.
In the technique scheme, if judge and contain lignin peroxidase specific coding genetic fragment in the solution to be measured, then can be according to the linear relationship between object chain mrna concentration and the galvanochemistry hybridization signal, set up equation of linear regression, measure the size of the concentration that contains lignin peroxidase specific coding genetic fragment in the solution to be measured, equation of linear regression can obtain by the following method:
Repeat the step of above-mentioned detection method, (2)～, (4), detect the hybridization solution of the Lip specific coding genetic fragment of a series of known variable concentrations, and according to the current-responsive result under each specific concentrations, (referring to Fig. 4), can draw the linear relationship between object chain mrna concentration and the galvanochemistry hybridization signal, can set up equation of linear regression, by experiment, the linear equation that draws is: DP%=, (3.4908 ± 0.1725) x+, (29.8172 ± 0.6892), (see figure 5), wherein, DP% is the electric current suppression ratio
I0 is the catalytic reduction electric current that object chain has neither part nor lot in competitive hybridization when reaction HRP, I
xThe catalytic reduction electric current of HRP when participating in the competition hybridization reaction for object chain; X is the natural logarithm of object chain concentration, and the range of linearity of concentration is 0.001 μ molL
-1～1 μ molL
-1, be limited to 1nmolL under detecting
-1The foundation that Lip specific coding genetic fragment concentration was calculated after this equation of linear regression can be used as.
Compared with prior art, the invention has the advantages that: the present invention is in conjunction with new bio molecule and nano material, with nano materials and chitosan-modified such as carbon nano-tube, magnetic nano-particle and golden nanometer particles in the carbon paste electrode surface, make up the electrochemical DNA sensor, greatly strengthen the electronic conduction ability of carbon paste electrode, thereby improve accuracy of detection; The DNA sensor, method that preparation of the present invention is complex film modified, with low cost, technology is simple; Utilize complex film modified DNA sensor of the present invention that Lip specific coding genetic fragment is detected, efficient, the low-cost dynamic change situation that detects gene of energy, thus the monitoring and the control procedure that can be the microbial degradation lignin provide a kind of effective biology tool.
Description of drawings
Fig. 1 is the structural representation of complex film modified DNA sensor of the present invention;
Fig. 2 is the composite membrane of complex film modified DNA sensor of the present invention and the assembling synoptic diagram of capture probe;
Fig. 3 is the hybridization reaction schematic diagram of complex film modified DNA sensor of the present invention;
Fig. 4 detects electric current-time changing curve figure that the Lip specific coding genetic fragment of variable concentrations obtains for the present invention makes up in the linear equation process with chronoamperometry.
Fig. 5 is the linear regression graph that Lip specific coding genetic fragment content of the present invention and electric current change.
Marginal data:
1, electric wire; 2, PTFE tube; 3, carbon-point; 4, magnet; 5, carbon paste; 6, sensitive materials (structure of composite membrane and DNA capture probe).
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment and accompanying drawing.
A kind of complex film modified DNA sensor as shown in Figure 1, comprise a carbon paste electrode, carbon paste electrode comprises carbon-point 3 and PTFE tube 2, carbon-point 3 is built in the PTFE tube 2, carbon-point 3 one ends are drawn PTFE tube 2 by electric wire 1, carbon-point 3 other ends contact with a magnet 4, magnet 4 is arranged at next-door neighbour's carbon-point 3 one ends and apart from the mouth of pipe 8mm place of PTFE tube 2, be filled with carbon paste 5 between the magnet 4 and PTFE tube 2 mouths of pipe, the induction end of carbon paste electrode is coated with sensitive materials 6, sensitive materials 6 comprise composite membrane and sulfhydrylation the DNA capture probe (sequence is 5 '-HS (CH
2)
6TTGTTGACGAAGGACTGCCA-3 '), composite membrane by modify magnetic nanoparticle successively in the sulfhydrylation on carbon paste electrode surface, the multi-walled carbon nano-tubes-golden nanometer particle and the shitosan of sulfhydrylation formed, the DNA capture probe is modified in multi-walled carbon nano-tubes-golden nanometer particle surface.
A kind of preparation method of above-mentioned complex film modified DNA sensor comprises following concrete steps:
(1) makes carbon paste electrode: in PTFE tube, put into carbon-point, be close to carbon-point one end and put into magnet apart from PTFE tube mouth of pipe 8mm place, form magnetic regions, fill with carbon paste between the magnet and the PTFE tube mouth of pipe, the carbon-point other end is drawn PTFE tube by electric wire, obtains the crude green body of carbon paste electrode, again with the surface finish of electrode crude green body, water flushing electrode surface is used HNO more successively then
3(massfraction is 50%), acetone, water carry out ultrasonic cleaning, again with the phosphate buffer flushing, dry naturally at last, obtain final carbon paste electrode;
(2) preparation magnetic nanoparticle and multi-walled carbon nano-tubes-golden nanometer particle composite membrane:
The technological process of preparation magnetic nanoparticle is: prepare Fe under nitrogen protection
3O
4Gelatinous precipitate adds polyglycol, positive silane ethyl ester, methyl alcohol, aminopropyl trimethoxysilane and halfcystine then successively, obtains the magnetic nanoparticle of sulfhydrylation after the reaction;
The technological process of preparation multi-walled carbon nano-tubes-golden nanometer particle is: use H
2O
2With sulfuric acid mixture liquid with the multi-walled carbon nano-tubes purifying, after the vacuum drying, with the reaction of itself and halfcystine, sulfydryl in modifications, multi-walled carbon nano-tubes and the golden nanometer particle with sulfhydrylation reacts again, obtains the multi-walled carbon nano-tubes-golden nanometer particle of sulfhydrylation
(3) decorating carbon paste electrode surface: the induction end surface of the magnetic nanoparticle for preparing and multi-walled carbon nano-tubes-golden nanometer particle and chitosan solution being modified successively the carbon paste electrode that step (1) makes, naturally obtain complex film modified carbon paste electrode after drying, again with the DNA capture probe of ready sulfhydrylation (sequence is 5 '-HS (CH
2)
6TTGTTGACGAAGGACTGCCA-3 ') drops in through complex film modified carbon paste electrode induction end surface, carry out self assembly, with the damping fluid flushing, obtain complex film modified DNA sensor after drying again by the sulfydryl and the effect of gold.
The DNA sensor that utilizes the foregoing description to make is 0.012 μ molL to concentration respectively
-1, 0.59 μ molL
-1With 0.22 μ molL
-1Lip specific coding genetic fragment testing sample measure, the detection of the Lip specific coding genetic fragment testing sample of each concentration is all carried out with the following step:
(1) choose object chain and modelled signal probe:
The sequence of choosing object chain is 5 '-TGGCAGTCCTTCGTCAACAA-3 '; According to the sequence of object chain, the sequence of modelled signal probe is 5 '-Biotin-TGGCAGTCCTTCGTCAACAA-3 ';
(2) competitive hybridization reaction: signal probe and object chain testing sample are splashed into the electrode surface of the DNA sensor that contains capture probe respectively, 37 ℃ of following competitive reactions 60 minutes;
(3) signalase amplifies: the phosphate buffered solution (pH 6.98) that will contain SA-HRP again drops in the DNA sensor surface that reacts through competitive hybridization, and 37 ℃ were reacted 30 minutes down, forms signalase connection amplification system;
(4) enzymic catalytic reaction: containing 1mmolL
-1Add 0.5mmolL in the phosphate buffer of p-dihydroxy-benzene (pH 7.38)
-1H
2O
2, with p-dihydroxy-benzene and H
2O
2Be substrate, with the DNA sensor that contains HRP signalase connection amplification system is working electrode, with saturated calomel electrode preferably as contrast electrode, with the platinized platinum electrode preferably as to electrode, set up the electrolytic cell of three-electrode system, adopt chronoamperometry, measure the DNA sensor under reduction potential-0.252V, the reduction current that HRP and substrate catalytic reaction produce changes that (electrochemical gaging is to adopt CHI660B electro-chemical systems that Shanghai occasion China instrument company produces to be connected with three-electrode system in the 50mL electrolytic cell, to control and to monitor), test result is as shown in table 1 below, by following table 1 as seen, the reduction current fall of three kinds of solution to be measured all is not less than 6.9%, all contains lignin peroxidase specific coding genetic fragment in three kinds of solution to be measured of decidable thus;
(5) quantitative test: according to reduction current fall measured in the following table 1, and in conjunction with equation of linear regression DP%=(3.4908 ± 0.1725) x+ (29.8172 ± 0.6892) that has made up, can determine Lip specific coding genetic fragment concentration value in three groups of testing samples, testing result sees the following form 1.
Table 1: the testing result of three kinds of variable concentrations testing samples
| Testing sample μ molL -1 | Reduction current fall % | Measure concentration μ molL -1 | Recovery % |
| 0.012 | 14.4 | 0.013±0.001 | 105.8 |
| 0.59 | 27.6 | 0.608±0.010 | 102.1 |
| 0.22 | 24.7 | 0.212±0.01 | 97.4 |
The result can clearly be seen that from said determination, this method is easy and simple to handle, and is highly sensitive, and selectivity is good, can be efficiently, low-cost online detection Lip specific coding genetic fragment content, for the monitoring and the control procedure of microbial degradation lignin provides technical support.
The above only is a preferred implementation of the present invention, and protection scope of the present invention also not only is confined to the foregoing description, and all technical schemes that belongs under the thinking of the present invention all belong to protection scope of the present invention.Should be pointed out that for those skilled in the art the some improvements and modifications not breaking away under the principle of the invention prerequisite all should be considered as protection scope of the present invention.
Claims (10)
1. complex film modified DNA sensor, comprise a carbon paste electrode, described carbon paste electrode comprises carbon-point and polyfluortetraethylene pipe, described carbon-point is built in the described polyfluortetraethylene pipe, described carbon-point one end is drawn described polyfluortetraethylene pipe by electric wire, the described carbon-point other end contacts with a magnet, be filled with carbon paste between the described magnet and the described polyfluortetraethylene pipe mouth of pipe, the induction end of described carbon paste electrode is coated with sensitive materials, it is characterized in that: described sensitive materials comprise the DNA capture probe of composite membrane and sulfhydrylation, described composite membrane is by modifying successively in the magnetic nanoparticle of the sulfhydrylation on described carbon paste electrode surface, multi-walled carbon nano-tubes-the golden nanometer particle of sulfhydrylation, and the shitosan composition, described DNA capture probe is modified in described multi-walled carbon nano-tubes-golden nanometer particle surface.
2. complex film modified DNA sensor according to claim 1 is characterized in that: described magnet is arranged at apart from the mouth of pipe of described polyfluortetraethylene pipe and is not more than the 8mm place.
3. complex film modified DNA sensor according to claim 1 and 2 is characterized in that: the sequence of described DNA capture probe is 5 '-HS (CH
2)
6TTGTTGACGAAGGACTGCCA-3 '.
4. the preparation method as claim 1 or 2 or 3 described complex film modified DNA sensors is characterized in that, comprises following processing step:
(1) makes carbon paste electrode: in polyfluortetraethylene pipe, put into carbon-point, and insert magnet at described carbon-point one end, form magnetic regions, fill with carbon paste between the described magnet and the polyfluortetraethylene pipe mouth of pipe, the carbon-point other end is drawn polyfluortetraethylene pipe by electric wire, obtain the crude green body of carbon paste electrode, more described crude green body is carried out surface treatment, obtain carbon paste electrode;
(2) decorating carbon paste electrode surface: the induction end surface of the magnetic nanoparticle for preparing and multi-walled carbon nano-tubes-golden nanometer particle and chitosan solution being modified successively the carbon paste electrode that step (1) makes, naturally obtain complex film modified carbon paste electrode after drying, DNA capture probe with ready sulfhydrylation drops in through complex film modified carbon paste electrode induction end surface again, after the self assembly, obtain complex film modified DNA sensor.
5. preparation method according to claim 4 is characterized in that, described magnetic nanoparticle is to prepare by following steps: prepare Fe under nitrogen protection
3O
4Gelatinous precipitate adds polyglycol, positive silane ethyl ester, methyl alcohol, aminopropyl trimethoxysilane and halfcystine then successively, obtains the magnetic nanoparticle of sulfhydrylation after the reaction.
6. preparation method according to claim 4, it is characterized in that, described multi-walled carbon nano-tubes-golden nanometer particle is to prepare by following steps: with the multi-walled carbon nano-tubes purifying, react with halfcystine again, make the multi-walled carbon nano-tubes sulfhydrylation, with the multi-walled carbon nano-tubes and the golden nanometer particle reaction of sulfhydrylation, obtain the multi-walled carbon nano-tubes-golden nanometer particle of sulfhydrylation then.
7. one kind is utilized complex film modified DNA sensor as claimed in claim 3 to the method that lignin peroxidase specific coding genetic fragment detects, and may further comprise the steps:
(1) choose object chain and modelled signal probe:
The sequence of choosing object chain is 5 '-TGGCAGTCCTTCGTCAACAA-3 '; According to the sequence of object chain, the sequence of modelled signal probe is 5 '-Biotin-TGGCAGTCCTTCGTCAACAA-3 ';
(2) competitive hybridization reaction: described signal probe and solution to be measured are splashed into the induction end surface of described complex film modified DNA sensor, and competitive reaction is no less than 60 minutes;
(3) signalase amplifies: be neutrality and the phosphate buffered solution that contains Avidin-horseradish peroxidase again with pH, drop in the induction end surface of the reacted DNA sensor of competitive hybridization, physiological temp reaction down is no less than 30 minutes, forms horseradish peroxidase signalase connection amplification system;
(4) enzymic catalytic reaction: at last with benzenediol and H
2O
2Be substrate, with the DNA sensor that contains described horseradish peroxidase signalase connection amplification system is working electrode, set up the electrolytic cell of three-electrode system, adopt the reduction current that produces in the chronoamperometry mensuration enzyme-catalyzed reaction to change, if the reduction current fall is not less than 6.9%, judge and contain lignin peroxidase specific coding genetic fragment in the solution to be measured, finish detection.
8. detection method according to claim 7, it is characterized in that, if judge and contain quality peroxidase specific coding genetic fragment in the solution to be measured, detect the concentration of the genetic fragment of lignin peroxidase specific coding described in the solution to be measured again according to following equation of linear regression;
Described equation of linear regression is: DP%=(3.4908 ± 0.1725) x+ (29.8172 ± 0.6892), wherein, DP% is the electric current suppression ratio, and x is the natural logarithm of described lignin peroxidase specific coding genetic fragment concentration, and the range of linearity that concentration detects is 0.001 μ molL
-1～1 μ molL
-1, be limited to 1nmolL under detecting
-1
9. according to claim 7 or 8 described detection methods, it is characterized in that: the three-electrode system in the described step (4) be with saturated calomel electrode as contrast electrode, with the platinized platinum electrode as to electrode.
10. according to claim 7 or 8 described detection methods, it is characterized in that: the reduction potential that detects in the described electrolytic cell for-0.3V～-0.2V, described electrolytic solution cell is the phosphate buffered solution of pH value for neutrality.
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