CN103743802A - DNA biosensor based on three-dimensional ordered gold doped nano titanium dioxide electrode as well as preparation method and application of DNA biosensor - Google Patents
DNA biosensor based on three-dimensional ordered gold doped nano titanium dioxide electrode as well as preparation method and application of DNA biosensor Download PDFInfo
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Abstract
The invention discloses a DNA biosensor based on a three-dimensional ordered gold doped nano titanium dioxide electrode and a preparation method as well as a method for detecting breast cancer genes by using the sensor. The preparation method comprises the following steps: preparing a three-dimensional ordered gold doped nano titanium dioxide film electrode (3DOMGTD/ITO) on an ITO (indium tin oxide) electrode by adopting a template method; then dropping a DNA probe (5'-Fc-ssDNA) labeled with ferrocene at 5' end on the electrode, thereby preparing a modified electrode, namely the DNA biosensor; placing the modified electrode in a solution of a breast cancer gene segment, hybridizing, detecting the variation of hybridization signals by adopting a differential pulse volt-ampere method, and carrying out electrochemical detection on the breast cancer gene. According to the invention, the label-type DNA biosensor is prepared by utilizing the specificity recognition effect between the DNA probe and the breast cancer gene, and the DNA biosensor has favorable stability, reproducibility and electrode regeneration, and can be applied to the field of DNA hybridization and tumor electrochemical detection.
Description
Technical field
The present invention relates to a kind of DNA biology sensor and preparation and application, relate in particular to a kind of marking type DNA biology sensor and preparation thereof based on three-dimensional order nano titanium oxide electrode, and this biology sensor is for the application in breast cancer tumour genetic test, belongs to DNA biosensor technology field and bioelectrochemistry technical field.
Background technology
Breast cancer is one of modal malignant tumour of women, is having a strong impact on even threat to life of women's physical and mental health.Research is found, in DNA molecular, the variation of base sequence is relevant with many genetic diseases of tumour and the mankind thereof, therefore research is carried out the identification of mastocarcinoma gene with specific DNA probe, quick, easy, the reliable detection method of development to mastocarcinoma gene, to diagnosing the early stage of breast cancer and treating to provide according to tool, be of great significance (referring to T.Ohmichi, Y. Kawamoto, P. Wu, D.Miyoshi, H.Karimata, N.Sugimot, Biochemistry, 2005,44,7125.).
In the detection method of breast cancer, although breast x-ray technology is effective method, yet for 40 years old following women because of the high detection sensitivity that makes of its mammary glandular cell density lower; It is fine that magnetic resonance imaging detects breast cancer sensitivity, but somewhat expensive and need the long period.Electrochemica biological sensor has that selectivity is good, highly sensitive, analysis speed is fast, required instrument is simple, can in complex system, carry out on-line continuous monitoring, be easy to realize the advantages such as microminiaturized, the fields such as clinical medical inspection and drug screening have been widely used in, for control, diagnosis and the treatment of human diseases have been opened up new approach (referring to Tiwari A, Gong S. Talanta, 2009,77 (3): 1217.).
Electrochemical DNA biosensor generally comprises four steps for measuring the process of DNA, as shown in Figure 1: (1) DNA probe is fixed: select suitable fixing means DNA probe to be fixed to the surface of electrode; (2) crossover process: the electrode that is fixed with DNA probe is placed in to solution to be measured, and under suitable condition, the target sequence in identification solution forms double-stranded DNA; (3) hybridization indication: hybridization reaction signal is converted into the electrochemical signals that can measure; (4) detection of electrochemical signals: electric current, voltage or electricity can be led as detection signal.
Nano material, with its high adsorption capacity, the advantageous property such as bio-compatibility is good, catalytic efficiency is high, is widely used in the immobilization technology of biomarker, amplifying signal and DNA in recent years.Nano particle possesses good biocompatibility, and the microenvironment of a similar biomolecule ontology environment can be provided for bioactive molecule, can effectively keep the activity of biomolecule; Nano particle has surface effect, makes it have higher adsorption ability, is the suitable medium of immobilizing biologically active molecule.Therefore, utilize nano particle as the carrier of fixing biological molecules, to build the active interface of biology sensor, can prepare well behaved biology sensor.
In the preparation of electrochemica biological sensor, bioactive molecule, in performances such as the stability that is fixedly directly connected to biology sensor of electrode surface, reappearance, sensitivity, is the committed step in preparation process.Ordered 3 D structure gold dopen Nano TiO
2both there is nano-TiO
2the performances such as distinctive optical, electrical, the catalysis presenting, possess again the good electric conductivity of nm of gold; In addition, ordered 3 D structure can make fixing biomolecule reach and align, is orientated regular object, thereby can effectively improve biomolecule at fixed efficiency and the electrochemical activity of electrode surface.
At present, be fixed on DNA probe on electrochemica biological sensor and be divided into two kinds, marking type and non-marked type.Compare with traditional non-marked type DNA probe, marking type probe not only can effectively reduce non-specific adsorption and background interference, can also improve sensitivity and the selectivity of identification and detection particular sequence DNA.The marking type DNA electrochemica biological sensor having developed at present passes through electroactive marker bioactive molecule mostly (referring to (a) Xiao Y; Qu X G, Kevin W. Plaxco., Alan J. Heeger. J. Am. Chem. Soc; 2007,129 (39): 11896; (b) Yang K, Zhang C Y. Anal. Chem, 2010,82 (22): 9500.), nano particle (referring to Zhang Y L, Wang Y, Wang H B, et al. Anal.chem., 2009,81:1982.), catalyzing enzyme (referring to Zhang J, Song S P, Zhang L Y, et al. J.Am.Chem.Soc., 2006, the sensitivity that 128:8575.) etc. mode improves analyzing and testing.The DNA probe of conventional electroactive label ferrocene (Fc) mark has good stability and redox reversible, because of but most widely used DNA electrochemica biological sensor labeled molecule (referring to (a) F.R.R.Teles, L.P.Fonseca. Talanta, 2008,77:606; (b) Sassolas A, Leca-Bouvier B D, Blum L.Chem.Rev., 2008,108 (1): 109; (c) Korri-Youssoufi H, Makrouf B. Anal Chim Acta, 2002,469: 85.).
Summary of the invention
The object of the invention is the DNA probe of ferrocene mark (5 '-Fc-ssDNA) to be fixed on nano titanium oxide electrode (3DOM GTD/ITO) surface of 3-D ordered multiporous structure, build a kind of new marking type DNA biology sensor, realize mastocarcinoma gene fragment (Breast-cancer
bRCA1 gene, ss1) Electrochemical Detection.
The present invention is achieved by the following technical solutions:
A DNA biology sensor based on three-dimensional order gold doped nano titanium dioxide electrode, is characterized in that, described biology sensor comprises the golden dopen Nano TiO of 3-D ordered multiporous structure
2the ITO electrode of modifying, its surperficial fixing DNA probe, described DNA probe is DNA probe 5 '-Fc-GAA CAA AAG GAA GAA AATC-(SH)-3 ' that 5 ' end is marked with ferrocene.
Described DNA biology sensor adopts following methods preparation, comprises the following steps:
1) prepare the golden dopen Nano TiO of 3-D ordered multiporous structure
2the ITO electrode of modifying, is labeled as 3DOM GTD/ITO modified electrode;
2) by the DNA probe of 5 ' end ferrocene mark, being labeled as 5 '-Fc-ssDNA solution drips in 3DOM GTD/ITO modified electrode surface, at 4 ℃, dry, make described DNA biology sensor, be labeled as 5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode.
Described 5 '-Fc-ssDNA solution is preferably 1.0 * 10
-55 ' of mol/L-Fc-ssDNA solution.
Described 3DOM GTD/ITO modified electrode adopts following methods preparation:
1) prepare golden dopen Nano TiO
2colloidal sol;
2) ITO handling well is placed at 1%PS bead (preferably 290nm) ethanolic solution, along with the volatilization of ethanolic solution, PS bead, by self-assembling to ITO electrode surface, forms six side's close-packed structures, then placed in baking oven in 95 ℃ of isothermal curings, obtained PS bead template;
3) PS bead template is tilted, pipette golden dopen Nano TiO
2colloidal sol drips along PS bead template inclined-plane, until infiltrate full wafer template, and the room temperature 5-10min that dries in the air; Again drip described golden dopen Nano TiO
2colloidal sol, repeats said process 3 times, dries standby;
4) in muffle furnace, the PS bead template in template is removed in high-temperature calcination, speed with 2 ℃/min is warming up to 500 ℃, and constant temperature 2h, then with the speed of 2 ℃/min, be down to room temperature, stay the golden dopen Nano TiO that porous ordered structure on ITO is ordered 3 D structure
2modified electrode (3DOM GTD/ITO).
The invention still further relates to the application of described DNA biology sensor, a kind of DNA biology sensor based on described detects the method for mastocarcinoma gene, comprises the following steps:
1) DNA probe fixing on modified electrode
Get 40 μ L 1.0 * 10
-5mol/L 5 '-Fc-ssDNA solution drips in 3DOM GTD/ITO modified electrode surface, at 4 ℃, dries, and makes 5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode;
2) hybridization reaction
Modified electrode described in step 1) is placed in to the 0.1mol/L phosphate buffer that comprises mastocarcinoma gene fragment ss1 hybridizes, described genetic fragment ss1 DNA sequence dna is 5 '-GAT TTT CTT CCT TTT GTTC-3 ', take out electrode, with redistilled water, clean, the genetic fragment ss1 that removes non-specific adsorption dries at 4 ℃;
Because the DNA probe 5 '-Fc-ssDNA on ss1 and modified electrode can match completely, form stable double helix chain, after hybridization, DNA probe peak current obviously reduces before hybridizing, and there is base mispairing in other genetic fragment and DNA probe, can not form stable double helix chain, therefore the DNA biology sensor in the present invention has specificity to ss1, and before and after hybridizing by 5 '-Fc-ssDNA and ss1, the variation of electrochemical signals can realize the Electrochemical Detection to breast cancer tumour gene.
3) Electrochemical Detection
Adopt three-electrode system with differential pulse voltammetry volt-ampere (DPV) method, to detect the variation of DNA hybridization signal, take respectively preparation 5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode and hybridization after this electrode be working electrode, saturated calomel electrode is contrast electrode, platinum filament is to electrode, at electrolytic solution, be in 0.1mol/L phosphate buffer (pH 7.0), to carry out differential pulse voltammetry volt-ampere (DPV) scanning, according to the changing value of peak current, measure the concentration of mastocarcinoma gene fragment ss1.
Particularly, the method for measuring the concentration of mastocarcinoma gene fragment ss1 in step 3) comprises the following steps:
A, the preparation of ss1 standard solution: prepare one group containing the phosphate buffered solution of different concentration known ss1;
B, set up working curve: the modified electrode of the DNA probe of preparation is immersed respectively in variable concentrations ss1 phosphate buffered solution and carries out hybridization reaction, after having reacted, electrode is taken out, with redistilled water, rinse, remove the not ss1 of hybridization, at 4 ℃, dry; This electrode after DNA probe electrode and hybridization is carried out to DPV scanning, the variation of recording peak current in phosphate buffered solution; Peak point current
iconcentration with ss1 in standard solution
cbe inversely proportional to, draw
i-
ctypical curve, or adopt linear regression method to obtain
i-
cequation of linear regression;
The detection of c, mastocarcinoma gene sample: testing sample is mixed with to solution, carries out hybridization reaction and the scanning of differential pulse voltammetry volt-ampere, recording responses electric current according to the method identical with step b in phosphate buffered solution; If response current is without significant change on the electrode after hybridization, show in testing sample not containing mastocarcinoma gene fragment ss1; If response current reduces, show in sample containing mastocarcinoma gene fragment, according to
i-
ctypical curve, obtains the concentration of ss1 in sample.
In described method, the temperature of hybridization reaction is 35～60 ℃, preferably 37 ℃.
In described method, the hybridization reaction time is >=1h.
In described method, the pH of the phosphate buffered solution that hybridization reaction is used in 5～8 scopes, preferably 6～7; Most preferably pH 7.0 PBS solution are as the damping fluid of hybridization reaction.
Compared with the prior art, the present invention has following beneficial effect:
Electrochemical DNA biosensor has that selectivity is good, highly sensitive, analysis speed is fast, required instrument is simple, can in complex system, carry out on-line continuous monitoring, be easy to realize the advantages such as microminiaturized.In the preparation of biology sensor, bioactive molecule, in performances such as the stability that is fixedly directly connected to biology sensor of electrode surface, reappearance, sensitivity, is the committed step in preparation process.Adopt at present Au dopen Nano TiO
2the method of modified electrode has been improved the supported quantity of biomacromolecule at electrode surface, but still exists immobilized stability to pay no attention to think of biomolecule and be difficult to the problems such as control in the immobilized orientation of electrode surface.
The present invention adopts three-dimensional order gold dopen Nano TiO
2modified electrode (3DOM GTD/ITO), 3-D ordered multiporous structure has the rigidity pore passage structure of mutual perforation and the specific surface area of superelevation, can improve well biomolecule in the immobilized effect of electrode surface, biomolecule supported quantity is large, immobilized condition is simple, adopt simple physisorption, and electrode material there are good biocompatibility and electric conductivity.Modified electrode has regular orderly three-dimensional structure, fixing biomolecule is reached and align, be orientated regular object, thereby can keep better the activity of biomolecule.
By SEM method, the pattern of the 3DOM GTD/ITO electrode surface of preparation is characterized the three-dimensional structure clear layer of 3DOM GTD/ITO electrode surface, in good order, aperture homogeneous.The DNA probe of ferrocene mark (5 '-Fc-ssDNA) is fixed on to electrochemical DNA biosensor prepared by electrode surface, can successfully detect breast cancer tumour gene, DNA probe presents linear relationship at the peak current of electrode surface and the concentration of breast cancer tumour gene, described sensor is quick, efficient, highly sensitive, and described method has lower detection limit, detect and be limited to 5.2 * 10
-7mol/L.
Below in conjunction with specific embodiment, describe the present invention.Protection scope of the present invention is not limited with embodiment, but is limited by claim.
Accompanying drawing explanation
Fig. 1 is the preparation of DNA electrochemica biological sensor and Electrochemical Detection process.
The three-dimensional order gold dopen Nano TiO of Fig. 2 for preparing at ITO electrode surface
2porous structure scanning electron microscope (SEM) photograph.
Fig. 3 is that Different electrodes is at Fe (CN)
6 3-/4-aC impedance figure in solution.(a), DNA probe modified electrode (curve b), DNA probe and control group DNA fragmentation hybridization modified electrode (curve c) and DNA probe and mastocarcinoma gene fragment are hybridized modified electrode (curve d) to curve to be respectively 3DOM GTD/ITO electrode.
Fig. 4 is on 3DOM GTD/ITO electrode, the differential pulse voltammetry volt-ampere curve of DNA probe in the mastocarcinoma gene fragment solution of variable concentrations, and wherein mastocarcinoma gene concentration a-g is respectively: 1 * 10
-5, 8 * 10
-6, 6 * 10
-6, 4 * 10
-6, 2 * 10
-6, 1 * 10
-6, 8 * 10
-7mol/L.
Fig. 5 is that DNA probe is in the peak current of electrode surface and the linear relationship of mastocarcinoma gene concentration.
Embodiment
Embodiment 1
30mL absolute ethyl alcohol is placed in to 100mL conical flask, adds while stirring 0.3mL triethanolamine, and then slowly add 0.7mL butyl titanate, continue to be stirred to solution and present the faint yellow of transparent and homogeneous, then add 10mL HAuCl
4, water and ethanol the mixed solution (HAuCl of 0.5g/L
40.5mL, three water 3mL, absolute ethyl alcohol 6.5mL), stirring 6h, solution presents successively faint yellow in reaction, micro-yellow, blush, pink, until be finally claret, is golden dopen Nano TiO
2colloidal sol.Transmission electron microscope experimental result shows, nano particle is evenly distributed, and mean grain size is less than 25nm.
ITO electrode used (50 mm * 1.2, mm * 10 mm) first soaks 30min in alkali lye, then ultrasonic half an hour in isopropyl alcohol, ethanol, distilled water successively, uses afterwards intermediate water drip washing slide 3 times, naturally dries.Be placed in the measuring cup (35mm * 70mm) that 10mL 1%PS bead (290nm) ethanolic solution is housed the ITO handling well is outstanding, place about 3 days at 30 ℃.Along with the volatilization of ethanolic solution, PS bead, by self-assembling to ITO electrode surface, forms six side's close-packed structures.Then placed in baking oven and within 2 hours, be cured in 95 ℃ of constant temperature, obtained PS bead template.Scanning electron microscope (SEM) photograph by PS bead template sees, PS bead is in ITO electrode surface marshalling, and uniform surface is smooth.PS bead template is tilted slightly, with pipettor, pipette golden dopen Nano TiO
2 colloidal sol 10 μ L, slowly drip along PS bead template inclined-plane, until infiltrate full wafer template, and the room temperature 5-10min that dries in the air; Again drip, repeat said process 3 times, dry standby.Adopt muffle furnace high-temperature calcination to remove the PS bead template in template, speed with 2 ℃/min is warming up to 500 ℃, and constant temperature 2h, then with the speed of 2 ℃/min, be down to room temperature, stay the golden dopen Nano TiO that porous ordered structure on ITO is ordered 3 D structure
2modified electrode (3DOM GTD/ITO),, aperture homogeneous regular in order by the three-dimensional structure of the visible preparation of scanning electron microscope (SEM) photograph (Fig. 2).
Embodiment 2
The 3DOM GTD/ITO electrode that embodiment 1 is made is rinsed well with intermediate water, and room temperature is dried.By 40 μ L1.0 * 10
-5mol/L 5 '-Fc-ssDNA drops in 3DOM GTD/ITO electrode surface, at 4 ℃, dries, and makes 5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode, and 4 ° of C preserve.
The UV-Vis absorption spectrum of 5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode has a DNA characteristics absorption peak at 260 nm places, shows that DNA probe has been fixed on 3DOM GTD/ITO electrode surface.On 5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode, the peak position of DNA and the DNA peak position of free state are almost consistent, show that the DNA probe being fixed on 3DOM GTD/ITO electrode still keeps its biologically active.
DNA probe molecule can produce obvious electrochemical signals at 3DOM GTD/ITO electrode surface, there is a pair of obvious redox peak at 0.460V and 0.307V in the cyclic voltammetry curve of 5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode, this is that Fc by DNA probe molecule produces in electrode surface generation redox reaction to peak.Because the potential difference (PD) at redox peak is less than 200 mV, can be according to formula
k s=mnF/RT calculates the electron transfer rate constant of Fc on electrode
k sfor 4.87S
-1.Redox peak current increases along with sweeping fast increase peak point current, redox peak current with sweep rapid-result linear relationship, show that this reaction velocity is subject to the surface electrochemistry process control of electrode.
Embodiment 3
The present embodiment utilizes the interactional principle of specificity of DNA probe (5 '-Fc-ssDNA) and two complementary DNA single interchains of mastocarcinoma gene fragment (ss1), significant change by DNA probe peak current before and after hybridization detects ss1, realizes the Electrochemical Detection to breast cancer tumour gene.
5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode is placed in to the solution of 2mL ss1, at 37 ℃, hybridizes 1 h.Take out electrode, with redistilled water, fully clean, to remove the ss1 of non-specific adsorption, at 4 ℃, dry.Alternative is selected a random genetic fragment ss2 and is tested as a control group, and ss2 DNA sequence dna is: 5 '-GGT CAG GTG GGG GGT ACG CCA GG-3 '.
Electrochemical Detection adopts three-electrode system, and take 5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode of preparing and this electrode after hybridization is working electrode, and saturated calomel electrode is contrast electrode, and platinum filament is to electrode.While carrying out cyclic voltammetric (CV) and differential pulse voltammetry volt-ampere (DPV) measurement, electrolytic solution is 0.1mol/L phosphate buffer (pH 7.0).Before experiment, pass into high-purity N
215min deoxygenation, experiment is at N
2under protection, carry out.All experimental temperatures are 25 ℃.
While there is ss1 in solution, DNA probe and the ss1 differential pulse voltammetry volt-ampere experimental result on 3DOM GTD/ITO electrode shows, obviously reduces with the electric current of DNA probe after ss1 hybridization reaction.Because ss1 and DNA probe can match completely, form stable double helix chain, DNA probe electric current is obviously reduced; And there is base mispairing with DNA probe in control group DNA fragmentation (ss2), can not form stable double helix chain, make DNA probe electric current that significant change not occur.Because DNA molecular is non-conductive, when electrode surface is fixed with after DNA probe, make Fe (CN)
6 3-/4-hindered with the electronics transfer of electrode surface, it is large that the resistance value of electrode surface becomes.When DNA probe and ss1 hybridization forms after duplex structure at electrode surface, the electroactive group Fc that comparatively duplex structure of rigidity makes DNA probe 5 '-end is with electrode surface apart from becoming greatly, and the resistance value of electrode surface further increases.The resistance variations on AC impedance measuring Different electrodes surface, as shown in Figure 3, further proves after DNA probe and mastocarcinoma gene fragment ss1 hybridization, the transmission of electrode surface electronics is obstructed, thereby the electric current of DNA probe obviously reduces.
Embodiment 4
Method is with embodiment 3, and difference is to change the temperature of hybridization reaction.
In DNA probe and mastocarcinoma gene crossover process, the change of hybridization temperature affects the degree of hybridization reaction, and when hybridization temperature is too low, the DNA probe diffusion in solution slowly, will affect the pairing between complementary strand base; During excess Temperature, can cause DNA sex change.When temperature is during lower than 35 ℃, almost do not observe the generation of hybridization reaction in this experiment.Temperature in the scope of 35～60 ℃ time, can detect obvious DNA probe electrochemical signals and change.Recommendations for selection hybridization temperature of the present invention is 37 ℃.
Method is with embodiment 3, and difference is to change the time of hybridization reaction.
In DNA probe and mastocarcinoma gene crossover process, the degree of hybridization reaction is the function of time.Along with the increase of hybridization time, the degree of hybridization reaction also increases, and the peak current that DNA probe produces reduces.The present embodiment shows, after hybridization reaction 1 h, DNA probe peak current tends towards stability, and shows that hybridization reaction tends to balance, so the time of recommendations for selection DNA probe of the present invention and mastocarcinoma gene hybridization reaction is 1 h.
Embodiment 6
Method is with embodiment 3, and difference is to change the pH of hybridization reaction phosphate buffered solution used.
The pH of solution also can affect DNA probe electrochemical signals in hybridization reaction, and when pH>7.0, the redox peak of DNA probe reduces along with the increase of pH, and when pH>8.0, redox peak almost disappears.In conjunction with the physiological environment of human body, recommendations for selection pH 7.0 PBS solution of the present invention are as the damping fluid of hybridization reaction.
Embodiment 7
NDA probe modification electrode 5 '-Fc-ssDNA/3DOM GTD/ITO of take is working electrode, adopt method of the present invention to detect mastocarcinoma gene, while containing mastocarcinoma gene fragment ss1 in solution, variation by differential pulse voltammetry volt-ampere sweep record Fc peak current, can realize the Electrochemical Detection to mastocarcinoma gene.
A, ss1 standard solution preparation: to the aqua sterilisa that adds respectively 56 μ L in 5.6nmol ss1, obtaining concentration is 1.0 * 10
-4the ss1 storing solution of mol/L.Required variable concentrations solution dilution in proportion again in experiment, preparation is containing the phosphate buffered solution (hybridization solution, pH 7.0) of variable concentrations ss1, and solution is placed in 4 ° of C Refrigerator stores;
B, set up working curve: the modified electrode of the DNA probe of preparation is immersed respectively in variable concentrations ss1 phosphate buffered solution and carries out hybridization reaction 1h, after having reacted, electrode is taken out, with redistilled water, rinse 3 times, remove the not ss1 of hybridization, at 4 ℃, dry.This electrode after DNA probe electrode and hybridization is carried out to DPV scanning in pH 7.0 phosphate buffered solution, record the variation of Fc peak current, as shown in Figure 4.The peak point current of Fc
iconcentration with ss1 in standard solution
cbe inversely proportional to, draw
i-
ctypical curve, adopts linear regression method to obtain
i-
cequation of linear regression;
When the mastocarcinoma gene ss1 of variable concentrations is hybridized with the DNA probe of electrode surface respectively, the peak point current of Fc
iconcentration with ss1 in solution
cbe inversely proportional to, differential pulse voltammetry experimental result shows: along with the increase (Fig. 4, g-a) of ss1 concentration, Fc peak point current reduces gradually.When ss1 concentration is 8.0 * 10
-7mol/L ~ 1.0 * 10
-5in the scope of mol/L, the peak point current of Fc and the concentration of ss1 present linear relationship.
i-
ctypical curve, as shown in Figure 5.Linear equation is: y (μ A)=0.5005 * 10
5x (μ mol/L)-0.9577, linearly dependent coefficient is 0.9908, detects and is limited to 5.2 * 10
-7mol/L (S/N=3).
Embodiment 8
By DNA probe modified electrode continuous circulation scanning 40 circles under the sweep velocity of 100mV/s, redox spike potential is almost constant, and peak current slightly reduces.On 4 3DOM GTD/ITO electrodes of preparing respectively, modify the DNA probe of same amount, carry out respectively scan round, the redox spike potential obtaining is consistent, and the relative standard deviation of peak current is less than 5%, shows that prepared modified electrode has good reappearance.
Claims (8)
1. the DNA biology sensor based on three-dimensional order gold doped nano titanium dioxide electrode, is characterized in that, described biology sensor comprises the golden dopen Nano TiO of 3-D ordered multiporous structure
2the ITO electrode of modifying, its surperficial fixing DNA probe, described DNA probe is DNA probe 5 '-Fc-GAA CAA AAG GAA GAA AATC-(SH)-3 ' that 5 ' end is marked with ferrocene, is labeled as 5 '-Fc-ssDNA.
2. a preparation method for DNA biology sensor claimed in claim 1, comprises the following steps:
1) prepare the golden dopen Nano TiO of 3-D ordered multiporous structure
2the ITO electrode of modifying, is labeled as 3DOM GTD/ITO modified electrode;
2) DNA probe solution is dripped in 3DOM GTD/ITO modified electrode surface, described DNA probe is DNA probe 5 '-Fc-GAA CAA AAG GAA GAA AATC-(SH)-3 ' of 5 ' end ferrocene mark, is labeled as 5 '-Fc-ssDNA; At 4 ℃, dry, make described DNA biology sensor, be labeled as 5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode.
3. the preparation method of DNA biology sensor according to claim 2, is characterized in that, described 3DOM GTD/ITO modified electrode adopts following methods preparation:
1) prepare golden dopen Nano TiO
2colloidal sol;
2) ITO handling well is placed at 1%PS bead ethanolic solution, along with the volatilization of ethanolic solution, PS bead, by self-assembling to ITO electrode surface, forms six side's close-packed structures, then placed in baking oven in 95 ℃ of isothermal curings, obtained PS bead template;
3) PS bead template is tilted, pipette golden dopen Nano TiO
2colloidal sol drips along PS bead template inclined-plane, until infiltrate full wafer template, and the room temperature 5-10min that dries in the air; Again drip described golden dopen Nano TiO
2colloidal sol, repeats said process 3 times, dries standby;
4) in muffle furnace, the PS bead template in template is removed in high-temperature calcination, with the speed of 2 ℃/min, is warming up to 500 ℃, and constant temperature 2h, then with the speed of 2 ℃/min, is down to room temperature, makes described 3DOM GTD/ITO modified electrode.
4. based on DNA biology sensor claimed in claim 1, detect a method for mastocarcinoma gene, comprise the following steps:
1) DNA probe fixing on modified electrode
Get 40 μ L 1.0 * 10
-5mol/L 5 '-Fc-ssDNA solution drips in 3DOM GTD/ITO modified electrode surface, at 4 ℃, dries, and makes 5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode;
2) hybridization reaction
Modified electrode described in step 1) is placed in to the 0.1mol/L phosphate buffer that comprises mastocarcinoma gene fragment ss1 hybridizes, described genetic fragment ss1 DNA sequence dna is 5 '-GAT TTT CTT CCT TTT GTTC-3 ', take out electrode, with redistilled water, clean, the genetic fragment ss1 that removes non-specific adsorption dries at 4 ℃;
3) Electrochemical Detection
Adopt three-electrode system with differential pulse voltammetry volt-ampere method, to detect the variation of DNA hybridization signal, take respectively preparation 5 '-Fc-ssDNA/3DOM GTD/ITO modified electrode and hybridization after this electrode be working electrode, saturated calomel electrode is contrast electrode, platinum filament is to electrode, at electrolytic solution, be in 0.1mol/L phosphate buffer, to carry out the scanning of differential pulse voltammetry volt-ampere, according to the changing value of peak current, measure the concentration of mastocarcinoma gene fragment ss1.
5. the method for detection mastocarcinoma gene according to claim 4, is characterized in that, the method for measuring the concentration of mastocarcinoma gene fragment ss1 in step 3) comprises the following steps:
A, the preparation of ss1 standard solution: prepare one group containing the phosphate buffered solution of different concentration known ss1;
B, set up working curve: the modified electrode of the DNA probe of preparation is immersed respectively in variable concentrations ss1 phosphate buffered solution and carries out hybridization reaction, after having reacted, electrode is taken out, with redistilled water, rinse, remove the not ss1 of hybridization, at 4 ℃, dry; This electrode after DNA probe electrode and hybridization is carried out to DPV scanning, the variation of recording peak current in phosphate buffered solution; Peak point current
iconcentration with ss1 in standard solution
cbe inversely proportional to, draw
i-
ctypical curve, or adopt linear regression method to obtain
i-
cequation of linear regression;
The detection of c, mastocarcinoma gene sample: testing sample is mixed with to solution, carries out hybridization reaction and the scanning of differential pulse voltammetry volt-ampere, recording responses electric current according to the method identical with step b in phosphate buffered solution; If response current is without significant change on the electrode after hybridization, show in testing sample not containing mastocarcinoma gene fragment ss1; If response current reduces, show in sample containing mastocarcinoma gene fragment, according to
i-
ctypical curve, obtains the concentration of ss1 in sample.
6. according to the method for the detection mastocarcinoma gene described in claim 4 or 5, it is characterized in that step 2) in, the temperature of hybridization reaction is 35～60 ℃.
7. according to the method for the detection mastocarcinoma gene described in claim 4 or 5, it is characterized in that step 2) in, the hybridization reaction time is >=1h.
8. according to the method for the detection mastocarcinoma gene described in claim 4 or 5, it is characterized in that step 2) in, the pH of the phosphate buffered solution that hybridization reaction is used is in 5～8 scopes.
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| CN104897759A (en) * | 2015-06-26 | 2015-09-09 | 彭梓 | Method for modifying indium tin oxide electrochemiluminescence reaction electrodes |
| CN105021676A (en) * | 2015-07-07 | 2015-11-04 | 河海大学 | Titanium dioxide microsphere modified electrode with three-dimensional dandelion-like hierarchical structure, and preparation method and application thereo |
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| CN108226259A (en) * | 2017-12-19 | 2018-06-29 | 北京科技大学 | The super infiltration high sensitive electrochemical microchip of one kind, preparation and application |
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| CN114574554A (en) * | 2022-02-23 | 2022-06-03 | 宁德师范学院 | TiO 22Defect-controlled DNA methylation photoelectric detection method |
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