CN102286371A - Alternating current impedance type deoxyribonucleic acid (DNA) electrochemical sensor based on probe DNA control assembly interface - Google Patents
Alternating current impedance type deoxyribonucleic acid (DNA) electrochemical sensor based on probe DNA control assembly interface Download PDFInfo
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- CN102286371A CN102286371A CN2011101893908A CN201110189390A CN102286371A CN 102286371 A CN102286371 A CN 102286371A CN 2011101893908 A CN2011101893908 A CN 2011101893908A CN 201110189390 A CN201110189390 A CN 201110189390A CN 102286371 A CN102286371 A CN 102286371A
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Abstract
The invention discloses an alternating current impedance type deoxyribonucleic acid (DNA) electrochemical sensor based on a probe DNA control assembly interface, which comprises an electrode and capture probe DNA, wherein the electrode adopts a gold electrode, and the capture probe DNA adopts sulfydryl modified DNA. The alternating current impedance type DNA electrochemical sensor is characterized in that the sulfydryl modified DNA is modified onto the surface of the gold electrode through the chemical bonding effect of Au-S bonds with the gold electrode at the room temperature and the absorption effect of the probe basic group parts and the gold so that the capture probe DNA flatly lies on the surface of the gold electrode for forming a capture probe DNA assembly layer, and bovine serum albumin exists on the surface of the capture probe DNA assembly layer and is used as sealing agents and protection agents. The impedance change before and after the hybridization is used as indication signals, the method adopts the surface assembly chemical technology for building the flat lying type DNA probe identification interface, simultaneously, the form of the probe maintains the flat lying state on the surface of the electrode without being influenced by the blank hybrid condition, and the method has the characteristics of high sensitivity, good specificity and the like.
Description
Technical field
The present invention relates to preparation method, belong to the biosensor technique field based on the alternating-current impedance type DNA electrochemical sensor of DNA control assembled interface.
Background technology
AC impedence method is a process of utilizing electrode surface changes in resistance reaction electrode configuration of surface to change, with EIS STUDY by modified electrode, simulate with the Randle circuit, resulting Nyquist figure comprises semicircle and two parts of oblique line, wherein semi-circular portions is a high frequency region, is controlled by electron transfer process; Linear portion is a low frequency range, represents diffusion-controlled step.Can reflect the size of electrode surface electron transport speed by the variation of Nyquist figure, thereby understand the information of relative dna hybridization kinetics and electrode interface textural property.This method has reduced the non-specific adsorption that causes owing to adding beacon material in the enzyme process, and need not to use hybridization indicator, therefore receives increasing concern in the research of transmitter.
The factor that influences electrode surface electron transport resistance has two kinds, and a kind of is electrocharge effect, i.e. electrode surface electronegativity material increase cause with electrolyte solution in electronegative electroactive substance [Fe (CN)
6]
3-/4-The increase of repulsive interaction, electrode surface Ret value increases; Another kind is a channelling effect, promptly when the electrode surface assembled layers has hole, makes [the Fe (CN) in the electrolyte solution
6]
3-/4-Arrive electrode surface by pore channel easily, accelerated electron transfer rate, electrode surface Ret value reduces.
The principle and the testing process of traditional alternating-current impedance type DNA electrochemical sensor are: (1) is fixed to gold electrode surfaces by sulfydryl with the mode that combines with the low temperature self-assembly of gold with ssDNA probe, and except sulfydryl also have a spot of DNA base with the gold surface bonded this moment; (2) carry out the secondary assembling with sulfydryl hexanol (MCH) as encapsulant, MCH is in the carat (measure of the purity of gold) point that sealing exposes, and its terminal sulfydryl is fought for DNA base institute bonded carat (measure of the purity of gold) point, makes DNA chain disengaging electrode surface and is in axial relatively state; (3) when the dna probe assembled layers of electrode surface and target dna solution under the hybridization conditions hybridization takes place after, the DNA chain of electrode surface increases, the negative charge quantity that the phosphoric acid skeleton produces increases, and simultaneously sterically hinderedly also increases, to [Fe (CN)
6]
3-/4-Produce bigger inhibition Deng the negative charge electrochemical probe in the electron exchange of electrode surface, resistance R et increases.
Yet because the dna probe density of electrode surface is higher, traditional alternating-current impedance type DNA electrochemical sensor just exists tangible space steric effect and electrocharge effect before DNA hybridization, when detection target dna concentration is low, sterically hindered and the electric charge that is increased is very limited, change not obviously though the alternating-current impedance value of hybridization increases to some extent, the method sensitivity of making is not high.
The present invention has designed a kind of new method, utilizes the surface-assembled chemical technology to make up " lying low " type dna probe identification interface, by the metamorphosis before and after the DNA hybridization of control electrode surface target dna is detected, and has improved the sensitivity of method greatly.
Summary of the invention
The purpose of this invention is to provide and a kind ofly make up " lying low " type dna probe identification interface and corresponding highly sensitive alternating-current impedance type DNA electrochemical sensor and preparation method thereof based on the surface-assembled chemical technology, its principle and testing process are as shown in Figure 1.
To achieve these goals, the present invention by the following technical solutions, a kind of alternating-current impedance type DNA electrochemical sensor of the present invention based on DNA control assembled interface,
Comprise electrode andCapture probe DNA
, electrode adopts gold electrode,Capture probe DNA adopts the DNA of sulfydryl modification, the DNA that it is characterized in that sulfydryl modification at room temperature with
Gold electrodeChemical bonding effect by the Au-S key and probe base portion and the adsorption of gold are modified gold electrode surfaces and capture probe DNA are lain low form capture probe DNA assembled layers in gold electrode surfaces; Bovine serum albumin is arranged as encapsulant and protective material on the surface of capture probe DNA assembled layers.
The preparation method who controls the alternating-current impedance type DNA electrochemical sensor of assembled interface based on DNA of the present invention comprises the steps:
(1) " lie low " structure at type dna probe identification interface
According to document [1] report, the DNA of sulfydryl modification except sulfydryl base portion also can with the mutual effect of golden hair looks, wherein base relative with golden effect a little less than, but speed is very fast.According to this principle, strand capture probe DNA with sulfydryl modification, at room temperature assemble fast, chemical bonding effect and probe base portion by the Au-S key are modified gold electrode surfaces with the adsorption of gold with it, capture probe DNA lay low and formed capture probe DNA assembled layers in electrode surface this moment, a large amount of carat (measure of the purity of gold) points is occupied, and places iron cyanogen electricity to solution this assembling electrode that has capture probe DNA assembled layers, [Fe (CN)
6]
3-/4-Be difficult to arrive by capture probe DNA assembled layers the gold surface of assembling electrode, electron transfer rate is slower, alternating-current impedance value Ret very big (Fig. 2).
Document [1]: Tonya M. Herne, and Michael J. Tarlov, Characterization of DNA Probes Immobilized on Gold Surfaces, J. Am. Chem. Soc., 1997,119 (38), 8916-8920.
(2) sealing and the protection at dna probe identification interface
With bovine serum albumin (BSA) as encapsulant and protective material; the carat (measure of the purity of gold) point of sealing exposure is to reduce non-special absorption on the one hand; protect the dna probe assembled layers not to be subjected to the influence of other factors (as heating, pH etc.) and the state of keeping lying low on the other hand in electrode surface.
(3) hybridization and detection
Because behind dna probe and its complementary sequence generation hybridization, form inflexible dna double spirane structure, this moment, the DNA base broke away from electrode surface, double-spiral structure stands on electrode surface, expose originally by the occupied space of DNA chain, form the passage that ion can free in and out, electron transport speed significantly increases, and electrode Ret value significantly reduces.Because the width of DNA chain is much smaller with respect to its length, even therefore the variation of configuration takes place in the dna probe of minute quantity owing to hybridization, also can expose more gold surface site and produce the passage that the chemical probe ion of can powering frees in and out, detection sensitivity improves.If the row that check order can not be hybridized with dna probe, then the assembled layers of electrode surface does not have metamorphosis, and this moment, change in impedance value was not obvious.Therefore can realize identification and detection complementary and incomplementarity DNA chain by the stool and urine of change in impedance value.Utilize the present invention detected result such as Fig. 3 to DNA, shown in Figure 4.
(curve a) after capture probe and complete complementary base sequence are hybridized as can be seen from Fig. 3 A result, resistance value significantly reduces, illustrate that probe can be good at hybridizing the formation duplex DNA with its fully-complementary sequence, this moment, double-stranded DNA stood on electrode surface, in measuring impedance liquid, [Fe (CN)
6]
3-/4-Arrive electrode surface by the hole between the DNA layer fast and carry out redox reaction, electron transport takes place, the electrode surface resistance value reduces.When capture probe and the interaction of single base mismatch sequence (curve b), though the electrode surface impedance significantly reduces than blank signal, but the signal that produces with fully-complementary sequence has tangible increase, fully reflects single base mismatch sequence and the fully-complementary sequence difference on hybridization efficiency.The signal (curve d) of the artificial synthesized sequence of mispairing generation is then very approaching with blank signal (curve e) fully, illustrates that constructed DNA electrochemical sensor has good sequence-specific.In addition, investigated of the influence of long-chain sequence with salmon sperm dna to measurement result, experimental result only shows otherwise has serial correlation, the signal that long-chain DNA is produced (curve c) promptly with blank value (curve e) no significant difference, further proved the good selectivity of this method.
Utilize constructed alternating-current impedance type DNA electrochemical sensor that specific gene sequence is carried out detection by quantitative as can be seen from Fig. 4 result, within the specific limits, along with the increase of target dna concentration, resistance value reduces gradually.Complementary strand concentration is 1.0 * 10
-13～1.0 * 10
– 8In the M scope, the concentration logarithm of resistance value and DNA is linear, detects and is limited to 3.0 * 10
-14M.
The invention provides the method for detecting specificity based on the alternating-current impedance type DNA electrochemical sensor of DNA control assembled interface, the concentration that it is characterized in that target dna is 1.0 * 10
-13～1.0 * 10
– 8In the M scope, the linear logarithmic function relation of the concentration of resistance value and DNA detects and is limited to 3.0 * 10
-14M.
Alternating-current impedance type DNA electrochemical sensor based on DNA control assembled interface of the present invention is used for identification form base mispairing sequence and fully-complementary sequence.
Advantage of the present invention is: with the single-stranded probe DNA of sulfydryl modification at room temperature with
Gold electrodeAssemble fast, chemical bonding effect and probe base portion by the Au-S key are modified gold electrode surfaces with the adsorption of gold with it, capture probe DNA lay low and formed capture probe DNA assembled layers in gold electrode surfaces this moment, a large amount of carat (measure of the purity of gold) points is occupied, place iron cyanogen electricity to solution this assembling electrode that has capture probe DNA assembled layers, [Fe (CN)
6]
3-/4-Be difficult to arrive by capture probe DNA assembled layers the gold surface of assembling electrode, electron transfer rate is slower, and alternating-current impedance value Ret is very big, sees the alternating-current impedance figure at the dna probe identification interface of Fig. 2.On the surface of capture probe DNA assembled layers bovine serum albumin (BSA) encapsulant and protective material are arranged; Behind the target dna generation hybridization of capture probe DNA and its complementary sequence, form the dna double spirane structure that inflexible stands on gold electrode surfaces, the impedance in the solution is reduced at iron cyanogen electricity.Above-mentioned has the susceptibility of height based on the constructed impedance type electrochemical DNA transmitter of surface-assembled chemistry for the identification complementary sequence, and detectability reaches 3.0 * 10
-14M can carry out detection by quantitative to specific gene sequence, is used for identification form base mispairing sequence and fully-complementary sequence.
Description of drawings
Fig. 1 is the schematic diagram based on the alternating-current impedance type DNA electrochemical sensor of dna probe control assembled interface.
Wherein: the capture probe dna marker among the figure is 1, and target dna is labeled as 2, and bovine serum albumin (BSA) encapsulant and protective material are labeled as 3, and electrode is labeled as 4.
Fig. 2 is the alternating-current impedance figure at dna probe identification interface: (a) 25 ℃ of assemblings of naked gold electrode (b), 2 h.
Fig. 3 A is the alternating-current impedance figure after different with the 10 nM respectively sequence DNA hybridization of dna probe: complete complementary DNA (a), single base mismatch DNA(b), complete mismatched dna (c), salmon sperm dna (d), blank signal (e).
Fig. 3 B be dna probe respectively with the hybridization of different sequence DNAs after alternating-current impedance value histogram.
Fig. 4 A is the alternating-current impedance figure after dna probe and the hybridization of different concns target dna; Concentration from a to f is respectively 10 nM, 1 nM, 100pM, 10 pM, 1 pM, 100 fM.
Fig. 4 B is the log-linear graph of a relation (concentration range 10 nM ~ 100 fM) of resistance value and target dna concentration.
Embodiment
As shown in Figure 1, the alternating-current impedance type DNA electrochemical sensor based on dna probe control assembled interface of the present invention, comprise electrode,Capture probe DNA and bovine serum albumin
, the preferred gold electrode 4 of electrode,The single stranded DNA of capture probe DNA 1 preferred sulfydryl modification, the DNA of sulfydryl modification except sulfydryl base portion also can with the mutual effect of golden hair looks, wherein base relative with the effect of gold a little less than, but speed is very fast.With the single-stranded probe DNA of sulfydryl modification at room temperature with
Gold electrodeAssemble fast, chemical bonding effect and probe base portion by the Au-S key are modified gold electrode surfaces with the adsorption of gold with it, capture probe DNA 1 lies low and forms capture probe DNA assembled layers in gold electrode 4 surfaces at this moment, a large amount of carat (measure of the purity of gold) points is occupied, place iron cyanogen electricity to solution this assembling electrode that has capture probe DNA assembled layers, [Fe (CN)
6]
3-/4-Be difficult to arrive by capture probe DNA assembled layers the gold surface of assembling electrode, electron transfer rate is slower, and alternating-current impedance value Ret is very big, sees the alternating-current impedance figure at the dna probe identification interface of Fig. 2.On the surface of capture probe DNA assembled layers bovine serum albumin (BSA) encapsulant and protective material 3 are arranged; After hybridizations take place the target dna 2 of capture probe DNA 1 and its complementary sequence, form the dna double spirane structure that inflexible stands on gold electrode surfaces, the impedance in the solution is reduced at iron cyanogen electricity.
Embodiment 1:
Preparation process based on the alternating-current impedance type DNA electrochemical sensor of dna probe control assembled interface is as follows:
(1) gold electrode Piranha solution (30% H
2O
2Dense H with concentration 98%
2SO
4, mix with the volume ratio of 1:3) and ultrasonic 5 min, with deionized water ultrasonic cleaning 2 times, each 5 min use 0.3 μ m, 0.05 μ m Al then respectively
2O
3Be polished to minute surface with the mixture of water, use ethanol, distilled water ultrasonic cleaning successively.Ultrasonic good electrode is placed 0.5 M H
2SO
4Middle cyclic voltammetry scan cleans N to stable with distilled water
2It is stand-by to dry up the back;
(2) get 4 μ l dna probes (oligonucleotide is given birth to biotechnology company limited by treasured and synthesized) solution, drip and be coated onto through pretreated naked gold electrode surfaces, after room temperature is placed 2 h, wash electrode surface respectively with PBS washing lotion, distilled water, after nitrogen dries up electrode is placed the 0.5% BSA solution of 300 μ l to soak 15 min, clean with distilled water, nitrogen dries up standby.
Embodiment 2:
Alternating-current impedance type DNA electrochemical sensor based on dna probe control assembled interface is as follows to the detection step of target dna:
(1) 40 min are hybridized in the capture probe that is fixed on electrode surface of embodiment 1 acquisition and target dna (oligonucleotide is given birth to biotechnology company limited by treasured and synthesized) 37 ℃ of water-baths in hybridization buffered soln, form double-stranded DNA, PBS washing lotion (pH 7.4) flushing electrode surface with 10 mM, remove the not DNA chain of hybridization, to be measured with distilled water flushing back again;
(2) electrode that step (1) is made immerses the 4 mM K that contain 0.1 M KCl
3[Fe (CN)
6]/K
4[Fe (CN)
6] in (1:1) the solution, open circuit potential is an initial potential, range of frequency 10
5Hz～1.0Hz, record alternating-current impedance curve.Alternating-current impedance figure sees the curve a of Fig. 3 A.
Embodiment 3:
The preparation of alternating-current impedance type DNA electrochemical sensor and as follows to the detection step of PML/RAR alpha fusion gene:
(1) gold electrode Piranha solution (30% H
2O
2Dense H with concentration 98%
2SO
4, mix with the volume ratio of 1:3) and ultrasonic 5 min, with deionized water ultrasonic cleaning 2 times, each 5 min use 0.3 μ m, 0.05 μ m Al then respectively
2O
3Be polished to minute surface with the mixture of water, use ethanol, distilled water ultrasonic cleaning successively.Ultrasonic good electrode is placed 0.5 M H
2SO
4Middle cyclic voltammetry scan cleans N to stable with distilled water
2It is stand-by to dry up the back;
The DNA sequence capture probe of (2) 5 ' end marking sulfhydryls is: 5 '-SH-(CH
2)
6-T
10CTTCA GAACT GCTGC TCTGG GTCTC AATGG-3 ' is mixed with the solution of 1 μ M;
(3) get the capture probe dna solution of 4 μ l steps (2), drip the naked gold electrode surfaces that is coated onto through pretreated step (1), after room temperature is placed 2 h, wash electrode surface respectively with PBS washing lotion, distilled water, after nitrogen dries up electrode is placed the 0.5% BSA solution of 300 μ l to soak 15 min, clean with distilled water, nitrogen dries up standby;
(4) be fixed on the 37 ℃ of water-baths in hybridization buffered soln of the capture probe of electrode surface and target dna and hybridize 40 min, form double-stranded DNA, with PBS washing lotion (pH 7.4) the flushing electrode surface of 10 mM, remove the not DNA chain of hybridization, to be measured with distilled water flushing back again;
(5) electrode is immersed the 4mM K that contains 0.1 M KCl
3[Fe (CN)
6]/K
4[Fe (CN)
6] in (1:1) the solution, open circuit potential is an initial potential, range of frequency 10
5Hz～1.0Hz, record alternating-current impedance curve.Alternating-current impedance figure sees Fig. 4 A, and the relation of impedance and concentration is seen Fig. 4 B.
By the experimental data of above embodiment and accompanying drawing, the dna probe that can prove sulfydryl modification of the present invention at room temperature can with
Gold electrodeAssemble fast, chemical bonding effect and probe base portion by the Au-S key are modified gold electrode surfaces with the adsorption of gold with it, capture probe DNA 1 lies low and forms capture probe DNA assembled layers in gold electrode 4 surfaces at this moment, " lying low " promptly of the present invention type dna probe identification interface, and constructed dna control assembled interface according to this
Alternating-current impedance type DNA electrochemical sensor.
Claims (5)
1. alternating-current impedance type DNA electrochemical sensor based on DNA control assembled interface,
Comprise electrode andCapture probe DNA
, electrode adopts gold electrode (4),Capture probe DNA (1) adopts the DNA of sulfydryl modification, the DNA that it is characterized in that sulfydryl modification at room temperature with
Gold electrodeChemical bonding effect by the Au-S key and probe base portion and the adsorption of gold are modified gold electrode surfaces and capture probe DNA (1) are lain low form capture probe DNA assembled layers in gold electrode (4) surface; Bovine serum albumin is arranged as encapsulant and protective material (3) on the surface of capture probe DNA assembled layers.
2. the described preparation method who controls the alternating-current impedance type DNA electrochemical sensor of assembled interface based on DNA of claim 1 comprises the steps:
(1) " lie low " structure at type capture probe DNA identification interface
With the single-stranded probe DNA of sulfydryl modification as capture probe DNA, at room temperature with the adsorption of gold it is modified gold electrode surfaces by the chemical bonding effect and the probe base portion of Au-S key, capture probe DNA lay low and formed capture probe DNA assembled layers in electrode surface this moment;
(2) sealing and the protection at capture probe DNA identification interface
, be added on capture probe DNA assembled layers surface and the gold electrode surfaces as encapsulant and protective material with bovine serum albumin (BSA).
3. the method for detecting specificity of the described alternating-current impedance type DNA electrochemical sensor based on DNA control assembled interface of claim 1, it is characterized in that 1) behind the target dna generation hybridization of capture probe DNA and its complementary sequence, the base of capture probe DNA breaks away from electrode surface, form inflexible dna double spirane structure, described dna double spirane structure stands on electrode surface, expose originally by the occupied space of DNA chain, form the passage that ion can free in and out; 2) can not hybridize as the target dna and the capture probe DNA of check order row, then the capture probe DNA assembled layers of electrode surface does not have metamorphosis, and this moment, change in impedance value was not obvious, realized identification and detection to incomplementarity DNA chain.
4. according to the method for detecting specificity of claim 3 based on the alternating-current impedance type DNA electrochemical sensor of DNA control assembled interface, the concentration that it is characterized in that target dna is 1.0 * 10
-13～1.0 * 10
– 8In the M scope, the linear logarithmic function relation of the concentration of resistance value and DNA detects and is limited to 3.0 * 10
-14M.
5. the described alternating-current impedance type DNA electrochemical sensor based on DNA control assembled interface of claim 1 is used for identification form base mispairing sequence and fully-complementary sequence.
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| CN104988214A (en) * | 2015-06-03 | 2015-10-21 | 福建医科大学 | Method for detecting acute promyelocyte PML/RAR alpha genetic sequence through double-electrode sensor based on multi-signal amplification technology |
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| CN105420090B (en) * | 2015-11-20 | 2020-05-05 | 青岛大学附属医院 | Gene detection system and method based on nanogold DNA probe |
| CN109610007A (en) * | 2018-10-12 | 2019-04-12 | 深圳市瀚海基因生物科技有限公司 | A kind of DNA chip and preparation method thereof that albumen is co-modified |
| CN109856217A (en) * | 2019-03-07 | 2019-06-07 | 广西师范学院 | Method based on electrochemical AC impedance detection miRNA-21 |
| CN109856217B (en) * | 2019-03-07 | 2021-10-15 | 宁波远志立方能源科技有限公司 | Method for detecting miRNA-21 based on electrochemical alternating current impedance |
| WO2023035609A1 (en) * | 2021-09-10 | 2023-03-16 | 常州先趋医疗科技有限公司 | Rapid gene screening method and device |
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