CN113584129B - P53 gene detection probe, obtained biosensor and application thereof - Google Patents

P53 gene detection probe, obtained biosensor and application thereof Download PDF

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CN113584129B
CN113584129B CN202110870000.7A CN202110870000A CN113584129B CN 113584129 B CN113584129 B CN 113584129B CN 202110870000 A CN202110870000 A CN 202110870000A CN 113584129 B CN113584129 B CN 113584129B
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porous hollow
magnetic
particles
gene detection
detection probe
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CN113584129A (en
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李昉
张小凡
于潇潇
李坤浩
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Qingdao University of Science and Technology
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a p53 gene detection probe, an obtained biosensor and application thereof, and relates to the field of biosensors. The p53 gene detection probe provided by the invention consists of porous hollow magnetic nano-particles, electrochemical redox active probes encapsulated in the porous hollow magnetic nano-particles, a cationic polymer functional layer coated on the surfaces of the porous hollow magnetic nano-particles and capture DNA adsorbed on the surfaces of the functional layer. The biosensor provided by the invention combines the advantages of electrochemistry, enzyme digestion circulation and magnetic materials, greatly reduces the requirements on instruments and test environments, improves the specificity, sensitivity, biocompatibility and detection limit, can be used as a powerful tool for p53 diagnosis, and has great potential.

Description

P53 gene detection probe, obtained biosensor and application thereof
Technical Field
The invention relates to the field of biosensors, in particular to a p53 gene detection probe, an obtained biosensor and application thereof.
Background
The oncogene p53 is involved in many biological processes, such as: plays an important role in regulating the biological functions of cell cycle, apoptosis, cell differentiation, DNA repair, aging and the like (Vousden K H and the like 2007). The p53 oncogene encodes and expresses p53 protein, inhibits malignant transformation of cells, and has a general antitumor effect, which is called "genome daemon" (Chen F et al 2010). Thus, sequence-specific analysis of the p53 gene plays a vital role in early screening and diagnosis of cancer. In response to the growing need for early diagnosis of cancer, researchers have developed many detection methods such as Quartz Crystal Microbalance (QCM), MALDI-TOF mass spectrometry, surface Plasmon Resonance (SPR), and colorimetry. Each method, although improved in some respects, has limitations in terms of ultra-micro sample detection, ease of handling, sensitivity, etc.; has the advantages of rapid detection, low cost and low sensitivity (Philip L Ross et al 1997; zhou W et al 2014).
Recent years have seen widespread use of electrochemical techniques for detecting ultra-low concentrations of DNA due to significant advantages, including high sensitivity, simplicity of operation, microliter sample volumes, low cost, simplicity, and portability, and electrochemical methods are expected to achieve ultra-sensitive detection of targets based on a variety of target amplification and signal amplification strategies (Wang Z et al 2016). CN 104031996a, after hybridized with target DNA by fixing a labeled DNA probe on the surface of the prepared nano gold particle, can form a new double strand with the fluorescent labeled duplex DNA under the catalysis of Taq DNA polymerase, so as to detect whether p53 gene mutation exists or not by fluorescence phenomenon. Although single base detection is possible, the limit of detection of this method is not reported. The sensor of p53 gene is prepared by electrode modified by chitosan-graphene, such as Zonghua Wang, gold nanoparticle-target DAN probe is fixed on the sensor, the specific surface area is greatly improved, the electrochemical signal can be obviously amplified, and the detection limit can reach 3.0x10 -16 M (Zonghua Wang et al 2016). Yang Liu et al prepared an electrochemiluminescence biosensor with enhanced surface plasmon resonance. The sensor uses gold nanoparticle @ polydopamine @ CuInZnS quantum dot nanocomposite. The oncogene p53 is detected in the amplified ECL detection system. The detection limit can reach 3.0X10 -8 M (Yang Liu et al 2018).
In electrochemistry, alkanethiol self-assembled monomolecular films on gold surfaces have been widely used in the preparation and adjustment of electrode/solution interface microenvironment and in the study of electron transfer. The monolayer of alkanethiol immobilized on the gold electrode has the ability to prevent the entry of redox species from the solution phase into the electrode surface. Magnetic nanomaterials have attracted considerable attention due to their excellent properties, including excellent superparamagnetism, biodegradability, biocompatibility, physiological condition stability, and dimensional dependence, and the like, with potential applications in magnetic fluid, catalysis, biomedical diagnostics, magnetic resonance imaging, and drug delivery technologies (Yayun Hu et al 2018). Fe (Fe) 3 O 4 Is one of the most important magnetic materials. Due to the multifunction of the nano composite material, the biosensor can meet the requirements of quick response, high sensitivity and high selectivitySelectivity requirements. However, the preparation process is complex, the preparation process cannot be reused, and the clinical use of the preparation process is limited due to high cost.
Disclosure of Invention
The invention provides a p53 gene detection probe, an obtained biosensor and application thereof, wherein the obtained biosensor can comprehensively improve the stable repetition rate, sensitivity and easy operation of an electrochemical biosensor.
In order to fully achieve the aim, the invention provides a p53 gene detection probe which consists of porous hollow magnetic nano-particles, electrochemical redox active probes encapsulated inside the porous hollow magnetic nano-particles, a cationic polymer functional layer coated on the surfaces of the porous hollow magnetic nano-particles and capture DNA adsorbed on the surfaces of the functional layer, wherein the capture DNA is a partial complementary sequence of the p53 gene, and the sequence is 5'-TCTTCCAGTGTGATG-3'.
The gene detection probe provided by the scheme creatively combines the recognition probe and the signal probe into a whole, can specifically recognize the p53 gene through complementary pairing of the capture DNA and the target DNA, and can also participate in hybridization to form double chains through the capture DNA, so that the electrochemical redox active probe sealed inside the magnetic nano-particles can be released. In addition, the magnetic nano particles have magnetism which is not possessed by other nano particles, so that the magnetic nano particles can be enriched by using a simple magnet, and meanwhile, the sensitivity, the anti-interference capability, the detection limit and the like of the detection can be improved. Compared with the existing detection probes, the detection probes provided by the invention have the functions of specifically identifying targets and generating electric signals, and are high in specificity, greatly reduced in operation flow and lower in cost.
Preferably, the porous hollow magnetic nanoparticle is selected from the group consisting of Fe 3 O 4 、Fe(O)、MnFe 2 O 4 、CoFe 2 O 4 、NiFe 2 O 4 Any one of them;
the electrochemical redox active probe is selected from [ Fe (CN) 6 ] 3- Any one of hydrophilic C60 nanomaterial, fc;
the cationic polymer is selected from any one of polydiallyl dimethyl ammonium chloride, polyethyleneimine, polyvinyl amine, polyvinyl pyridine, polyphosphate and polysilicates.
In the above scheme, the porous hollow magnetic nanoparticle is preferably Fe 3 O 4 Because the method is more economical and easy to obtain, has good separation effect, biocompatibility and degradability; the electrochemically redox-active probe is preferably [ Fe (CN) 6 ] 3- Because of its good charge transfer capability, it can react with the oxidizing group to release electrons and form obvious electric signals. The process of forming the electric signal does not need any external electric field or energy input and is driven by the electrochemical reaction of the process. Meanwhile, the electrochemical redox active probe encapsulated in the magnetic nanoparticle releases an electric signal after the capture DNA and the target DNA specifically form hybrid molecules, and the electric signal is controlled by the formation amount of the capture DNA-target DNA hybrid double-strand DNA, so that the specificity and the sensitivity of detection are greatly improved; the cationic polymer is preferably PDDA because it is safe, nontoxic, readily soluble in water, nonflammable, strong in cohesion, stable, insensitive to pH changes, and resistant to high temperatures. In addition, PDDA molecules contain a large amount of amino groups and are coated on the surfaces of the magnetic nanoparticles, so that the surfaces of the magnetic nanoparticles have positive charges, the capture of DNA on the surfaces of the magnetic nanoparticles in the next step is facilitated, the hydrophilicity of a system can be improved by adopting PDDA, the system has extremely high specificity and biocompatibility, high sensitivity and specificity are shown, and the specificity, stability and multiple functions of the magnetic nanoparticles can be improved.
The invention provides a preparation method of the p53 gene detection probe according to the technical scheme, which comprises the following steps:
adding the dried porous hollow magnetic nano particles into a cationic polymer solution, and carrying out ultrasonic treatment to obtain a uniform suspension;
performing magnetic separation on the obtained suspension, and collecting a magnetic separation product to obtain porous hollow magnetic nano particles coated with cationic polymer;
mixing porous hollow magnetic nano particles coated with cationic polymer with electrochemical redox active probe at 22-28 ℃ for 6-8h at 100-120r, adding capture DNA in Tris-HCl buffer system, and mixing at 22-28 ℃ for 100-120r for 6-8h to obtain magnetic composite material;
and performing magnetic separation, washing and drying on the obtained magnetic composite material to obtain the p53 gene detection probe.
The invention provides a biosensor for p53 gene detection, which comprises the p53 gene detection probe according to the technical scheme.
Preferably, the electrode also comprises a modified electrode, wherein the modified electrode consists of a 12-mercaptan modified gold electrode and methylene blue enriched on the surface of the 12-mercaptan.
Preferably, the modified electrode is prepared by the following steps:
s1: gold electrode pretreatment
First using concentrated H 2 SO 4 Solution and 30% H 2 O 2 Treating gold electrode with solution, washing gold electrode with deionized water, and washing with Al 2 O 3 And (3) polishing the powder, sequentially carrying out ultrasonic treatment by using deionized water, ethanol and deionized water, and finally flushing the treated electrode by using deionized water and drying the electrode by using high-purity nitrogen for standby.
S2: gold electrode surface modification
Soaking the treated gold electrode in absolute ethanol containing 12-mercaptan at room temperature, and standing for reaction under the conditions of light shielding and room temperature; then, washing the electrode by absolute ethyl alcohol, and drying the surface of the gold electrode by high-purity nitrogen; and finally, modifying a compact 12-mercaptan monomolecular layer stable structure on the surface of the electrode.
S3: MB enrichment
The 12-mercaptan on the surface of the electrode has strong adsorption capacity to MB, and when the electrode modified with the 12-mercaptan is placed in MB solution, MB is rapidly enriched on the surface of the electrode. And taking out the electrode and washing to obtain the modified electrode.
In the above scheme, 12-thiol is fixed on the electrode surface by the way that the thiol at the chain end forms an Au-S bond with Au. The amount of 12-thiol molecules immobilized on the electrode surface is proportional to the reaction time. The longer the time, the more thiol is immobilized. When the amount of 12-thiol molecules immobilized on the electrode surface reaches a certain amount, more thiol molecules cannot be bound due to steric hindrance.
Preferably, the treated gold electrode is immersed in absolute ethanol containing 12-mercaptan for a fixed reaction time of 3-5 hours, preferably 5 hours.
In the scheme, the prepared monomolecular layer on the surface of the modified electrode is stable and has strong anti-pollution capability, so that the response repetition rate of the electric signal is still kept at 90% after the modified electrode is used for 13-15 times. Compared with the prior art, the modified electrode provided by the invention has the advantages of high stable repetition rate, simple preparation process and high yield, and highlights the great potential of the modified electrode in clinical use.
Preferably, when the modified electrode is used in a biosensor to detect the p53 gene, the detection process is as follows:
immersing the electrode with the surface modified with the monolayer and MB in Tris-HCl buffer solution for standby;
and when the detection is carried out, the modified electrode is taken out from the buffer solution and immersed into the buffer solution of the sample to be detected, so that the detection can be carried out.
Preferably, the Tris-HCl buffer pH is 7.4-7.8, which is a mixed solution containing Tris, 100-110mM HCl and 100-120mM NaCl at a concentration of 110-150 mM.
Preferably, the modified electrode is immersed in a buffer solution of a sample to be detected at a constant temperature, and the detection condition is 37+/-1 ℃.
The invention provides an application of the p53 gene detection probe in detecting p53 genes.
Preferably, in the application, the p53 gene detection probe is added into a sample to be detected and exonuclease, incubated at 37+/-1 ℃ and reacted for 1.5-2 hours, the change condition of an electric signal is detected, and the content of the p53 gene in the sample to be detected is quantitatively detected.
Preferably, the volume ratio of the added p53 gene detection probe, the sample to be detected and the exonuclease is 5:5:0.5, and the exonuclease is ExoIII.
The p53 gene detection probe used in the scheme integrates the recognition probe and the electrochemical redox active probe, the incubation and hybridization reactions are carried out simultaneously, a reaction system is not required to be changed, the steps and stages are not required, the conditions are mild, and the operation is extremely simple. The incubation and reaction time is 1.5-2h, preferably 2h. This is because the incubation time affects the progress of hybridization and cleavage cycle reactions, and the reaction reaches equilibrium in approximately 2 hours, so that too long a time does not improve the occurrence of signals or increase the sensitivity.
In the above scheme, when the p53 gene detection probe detects the p53 gene, target DNA in a sample to be detected is specifically hybridized with capture DNA on the surface of the functionalized magnetic nano-particles, so that the capture DNA is separated from the surface of the magnetic nano-particles, thereby [ Fe (CN) 6 ] 3- Released from the magnetic material. The double-stranded DNA obtained after hybridization has a flat end, and is triggered to be digested by ExoIII, the target DNA is completely released, and hybridization can be continued with the capture DNA on the magnetic material to form circulation. Finally, a single target DNA can be hybridized to capture DNA on multiple magnetic materials to control [ Fe (CN) 6 ] 3- Is released. Through a series of cyclic enzyme digestion reactions, isothermal cyclic amplification of signals is realized. Released [ Fe (CN) 6 ] 3- Can be captured by a gold electrode, realizes electron transfer with MB on the surface of the electrode, and generates an electric signal. Thus, in the presence of target DNA, the electrochemical signal is significantly increased, enabling sensitive detection of the target by the intensity of the electrical signal response.
The invention provides an application of the biosensor in p53 gene detection.
Preferably, when in use, the p53 gene detection probe is added into a sample to be detected and exonuclease, and incubated and reacted for 1.5-2 hours at the temperature of 37+/-1 ℃;
removing magnetic nano particles in the reaction system through magnetic force selection, immersing the modified electrode in the rest reaction system, detecting the change condition of an electric signal by utilizing MB reaction of an electrochemical redox active probe and the surface of the modified electrode at the temperature of 37+/-1 ℃, and quantitatively detecting the content of p53 genes in a sample to be detected.
Preferably, the p53 gene is a p53 gene in human serum.
Preferably, when the biosensor is used for detecting p53 gene in human serum, the detection process is as follows:
immersing the electrode with the surface modified with the monolayer and MB in Tris-HCl buffer solution for standby;
and diluting the human serum sample with Tris-HCl buffer solution, taking out the modified electrode from the buffer solution when the detection is carried out, and immersing the modified electrode into the buffer solution of the human serum sample to carry out the detection.
Preferably, the Tris-HCl buffer is Tris-HCl buffer with pH of 7.4-7.8, and is a mixed solution containing the concentration of 110-150mM of Tris, 100-110mM of HCl and 100-120mM of NaCl.
Preferably, the human serum sample is diluted to a concentration of 2%.
Compared with the prior art, the technical scheme of the invention has the advantages that:
1. the p53 gene detection probe provided by the invention integrates the identification probe and the signal probe, has integrated functions, is favorable for carrying out uniform electrochemical analysis on a target under the condition of not needing any fixed program, reduces the complexity of a sensor structure, has simple operation, high sensitivity and low cost, and is favorable for clinical use;
2. the p53 gene detection probe provided by the invention seals porous Fe by complementary DNA of p53 gene 3 O 4 Magnetic nanoparticles, specific hybridization of p53 gene with complementary DNA can open porous Fe 3 O 4 The magnetic nano particles release high-sensitivity charged particles and combine enzyme digestion circulation, so that isothermal circulation amplification of signals is realized, the detection limit (6.4 fM) is greatly reduced, and the specificity is improved.
3. The modified electrode provided by the invention adopts the monomolecular layer to modify the surface of the gold electrode, so that the anti-pollution capability of the electrode is greatly improved, and the response repetition rate of an electric signal can still be kept at 90% after the modified electrode is used for 13-15 times;
4. the biosensor provided by the invention is prepared from [ Fe (CN) 6 ] 3- Embedding in magnetic nanometer material, fe can be realized through simple magnetic field screening 3 O 4 Magnetic nanoparticle/[ Fe (CN) 6 ] 3- The rapid and efficient enrichment of the sensor avoids the interference of impurities in the next detection process and improves the sensitivity of the sensor.
Through the cooperative implementation of the four technical measures, the advantages of electrochemistry, enzyme digestion circulation and magnetic materials are combined together, the requirements on instruments and testing environments are greatly reduced, and the specificity, sensitivity, biocompatibility and detection limit are improved. The p53 gene detection probe/modified electrode/biosensor provided by the invention integrates the advantages of high electrode stability and repetition rate, high sensitivity, easiness in operation, enzyme catalytic amplification, wide detection range, strong electrode stability and the like, can be used as a powerful tool for p53 diagnosis, and has great potential.
Drawings
FIG. 1 shows Fe provided by the embodiment of the invention 3 O 4 A Transmission Electron Microscope (TEM) image of the magnetic nanoparticle, wherein (a) and (b) are low resolution TEM images at different magnifications, (c) a hollow block diagram of a single magnetic bead, (d) a high resolution TEM image;
fig. 2 is a Zeta potential plot of different nanoparticles provided by an embodiment of the present invention, wherein the substances represented by curves 1 to 4 are:
(1)Fe 3 O 4 magnetic nanoparticles, (2) PDDA/Fe 3 O 4 Magnetic nanoparticles, (3) [ Fe (CN) 6 ] 3- PDDA/Fe3O4NPs, (4) Capture DNA/[ Fe (CN) 6 ] 3- /PDDA/Fe 3 O 4 Magnetic nanoparticles;
FIG. 3 is a graph showing the response of an electrical signal of a modified electrode after multiple uses provided by an embodiment of the present invention;
FIG. 4 is a cyclic voltammogram of different concentrations of target DNA provided by an embodiment of the present invention, wherein the target DNA concentrations represented by curves 1 through 8 are, respectively: (1) 40nM, (2) 10nM, (3) 4nM, (4) 200pM, (5) 60pM, (6) 20pM, (7) 100fM, (8) 0.
FIG. 5 is a plot of SWV response for a no target and four different DNA sequences provided by an embodiment of the present invention, in order from top to bottom: complete hybridization matched Target DNA (TD), single base mismatched DNA (1 MT), two base mismatched DNA (2 MT), complete mismatched DNA (NC), and no target;
FIG. 6 is a graph of CV current response of target DNA at various concentrations in human serum, represented by curves 1 to 8, according to an embodiment of the present invention, wherein the concentrations of target DNA are: (1) 40nM, (2) 10nM, (3) 4nM, (4) 200pM, (5) 60pM, (6) 20pM, (7) 100fM, (8) 0.
Detailed Description
The invention will be further illustrated with reference to a few non-limiting examples in order to clarify the characteristics of the invention. All changes and modifications that may be made in accordance with the principles of the present invention after reading the teachings set forth herein are intended to be covered by the appended claims.
EXAMPLE 1Fe 3 O 4 Preparation of magnetic nanoparticles
Fe 3 O 4 The magnetic nanoparticles are prepared by a solution thermal method.
The specific process is as follows: 0.5g of ferric chloride, 1.5g of trisodium citrate and 0.5g of urea were completely dissolved in 40mL of distilled water, and the mixed solution was stirred at room temperature for 10min. 0.5g of polyacrylamide was dissolved in 40mL of deionized water to give a viscous polyacrylamide solution, which was slowly added to the vigorously stirred mixed solution. The mixture was then transferred to a 100mL teflon lined autoclave for sealing, heated to 200 ℃ and held for 12h. Magnetically separating the product obtained after the reaction by a magnetic frame, collecting the product, washing the product with distilled water and absolute ethyl alcohol for three times respectively, and finally drying the product at 60 ℃ for 3 hours for later use to obtain porous Fe serving as a material for experiments 3 O 4 Magnetic nanoparticles.
Fe prepared as can be seen from FIGS. 1 (a) and (b) 3 O 4 The magnetic nano particles have uniform particle size and average diameter of 250nm. (c) Fe can be seen in 3 O 4 The magnetic nanoparticles were bright in the center and darker in the periphery, indicating that the particles were hollow porous structures. (d) The Fe produced can be seen 3 O 4 The lattice of the magnetic nanoparticles was about 0.241nm.
Example 2: fe coating PDDA 3 O 4 Preparation of magnetic nanoparticles
Step (1) Fe 3 O 4 Preparation of magnetic nanoparticles: as in example 1.
Step (2) PDDA coating Fe 3 O 4 Magnetic nanoparticles.
The specific process is as follows: for 10mg of porous Fe taken out 3 O 4 The magnetic nanoparticles were added to 5mL of PDDA solution at a concentration of 30mg/mL, and sonicated for 0.5-1h. Then magnetic separation is carried out on the suspension by using a magnetic frame, and the magnetic separation product is collected and dried for 3 hours at 60 ℃ to obtain PDDA/Fe with positive charges 3 O 4 Magnetic nanoparticles.
Example 3: fe coating PDDA 3 O 4 Preparation of magnetic nanoparticles
Step (1) Fe 3 O 4 Preparation of magnetic nanoparticles: as in example 1.
Step (2) PDDA coating Fe 3 O 4 Magnetic nanoparticles.
The specific process is as follows: for 10mg of porous Fe taken out 3 O 4 The magnetic nanoparticles were added to 5mL of PDDA solution at a concentration of 20mg/mL and sonicated for 0.5-1h. Then magnetic separation is carried out on the suspension by using a magnetic frame, and the magnetic separation product is collected and dried for 3 hours at 60 ℃ to obtain PDDA/Fe with positive charges 3 O 4 Magnetic nanoparticles.
Example 4: [ Fe (CN) 6 ] 3- /PDDA/Fe 3 O 4 Preparation of magnetic nanoparticles
Step (1) Fe 3 O 4 Preparation of magnetic nanoparticles: as in example 1.
Step (2) PDDA coating Fe 3 O 4 Magnetic nanoparticles. As in example 2.
Step (3) [ Fe (CN) 6 ] 3- Packaging into PDDA/Fe 3 O 4 Magnetic nanoparticles.
The specific process is as follows: 2mg PDDA/Fe 3 O 4 The magnetic nanoparticles were dissolved in 500. Mu.L of 80mM [ Fe (CN) 6 ] 3- Placing the solution in a shaking table, maintaining the temperature at 25deg.C, revolution number at 100r, and time at 8h to obtain [ Fe (CN) 6 ] 3- /PDDA/Fe 3 O 4 Magnetic nanoparticles.
Example 5: [ Fe (CN) 6 ] 3- /PDDA/Fe 3 O 4 Preparation of magnetic nanoparticles
Step (1) Fe 3 O 4 Preparation of magnetic nanoparticles: as in example 1.
Step (2) PDDA coating Fe 3 O 4 Magnetic nanoparticles. As in example 2.
Step (3) [ Fe (CN) 6 ] 3- Packaging into PDDA/Fe 3 O 4 Magnetic nanoparticles.
The specific process is as follows: 2mg PDDA/Fe 3 O 4 The magnetic nanoparticles were dissolved in 500. Mu.L of [ Fe (CN) at a concentration of 60mM ] 6 ] 3- Placing the solution in a shaking table, maintaining the temperature at 25deg.C, revolution number at 100r, and time at 8h to obtain [ Fe (CN) 6 ] 3- /PDDA/Fe 3 O 4 Magnetic nanoparticles.
Example 6: capture DNA/[ Fe (CN) 6 ] 3- /PDDA/Fe 3 O 4 Preparation of magnetic nanoparticles
The specific process is as follows: mu.L of the captured DNA (0.08. Mu.M) was added to the above mixed solution, and the mixture was placed in a shaker at 25℃for 8 hours at 100 r. Subsequently, the magnetic composite material was magnetically separated, washed and resuspended in 500. Mu.L of 20mM Tris-HCl (pH 7.4, 100mM NaCl) solution using a magnetic rack.
Example 7: capture DNA/[ Fe (CN) 6 ] 3- /PDDA/Fe 3 O 4 Preparation of magnetic nanoparticles
The specific process is as follows: mu.L of the captured DNA (0.12. Mu.M) was added to the above mixed solution, and the mixture was placed in a shaker at 25℃for 8 hours at 100 r. Subsequently, the magnetic composite was magnetically separated, washed and resuspended in 500. Mu.L of 20mM Tris-HCl (pH 7.4, 100mM NaCl) solution using a magnetic rack.
As can be seen from FIG. 2, fe is produced 3 O 4 The magnetic nano-particles are negatively charged and the potential is negative; after capping with positively charged PDDA, fe 3 O 4 The magnetic nanoparticles showed a high positive zeta potential, indicating Fe 3 O 4 The magnetic nanoparticles are encapsulated by PDDA; then the redox probe [ Fe (CN) 6 ] 3- Packaging in the hole, reversing the potential to be negative; the capture DNA is wrapped in Fe by electrostatic action 3 O 4 The magnetic nanoparticle surface is more negative in potential. Wherein 1 corresponds to Fe 3 O 4 Zeta potential of magnetic nanoparticles (example 1); 2 correspond to PDDA/Fe 3 O 4 Zeta potential of composite nanoparticles (example 2); 3 corresponds to [ Fe (CN) 6 ] 3- /PDDA/Fe 3 O 4 Zeta potential of composite nanoparticles (example 4); 4 corresponding capture DNA [ Fe (CN) 6 ] 3- /PDDA/Fe 3 O 4 Zeta potential of composite nanoparticle (example 6). Examples 2, 4 and 6 were carried out as the best mode of practice and Zeta potential tests were carried out on the products prepared therefrom.
Example 8: preparation of modified electrode
The specific process is as follows:
at room temperature, the treated gold electrode was immersed in an absolute ethanol solution containing 1mM 1-Dodecanethiol, and allowed to stand still at room temperature for reaction for 5 hours under dark conditions. The electrode was then rinsed with absolute ethanol and the gold electrode surface was blown dry with high purity nitrogen.
Immersing the gold electrode prepared by the steps into MB solution, taking out after 5-10min, washing sequentially by absolute ethyl alcohol and deionized water, and preserving in deionized water for standby.
As can be seen from fig. 3, the response signal of the modified electrode remained 89% of the original response signal after 13 repeated uses. Therefore, the modified electrode prepared by the invention has good reproducibility and stability and has practical application value.
Example 9: correlation of target DNA with electric signals at different concentrations
To further verify the analytical detection capabilities of the electrochemical biosensors prepared by the present invention, target DNA (40 nM, 10nM, 4nM, 200pM, 60pM, 20pM, 100fM and blank samples) was detected at different concentrations of 0 to 40 nM. As can be seen from FIGS. 4, numbers 1-8 (corresponding concentrations from 40nM to blank), CV peak current increases with increasing target concentration, both positively correlated, i.e., higher target concentration, with Fe 3 O 4 The more capture DNA on the magnetic nanoparticle material binds, the more [ Fe (CN) 6 ] 3- The more released into the solution, the stronger the electrochemical signal can be detected.
Example 10: selectivity of biosensor for target DNA
To further verify the selectivity of the biosensor prepared by the present invention, by using the variation of the detection signal of the different DNA sequences including the perfectly complementary Target DNA (TD), the single base mismatched target (1 MT), the two base mismatched target (2 MT) and the non-complementary DNA (NC), the corresponding samples are respectively, from top to bottom, labeled 1 to > 5: full complement Target DNA (TD), single base mismatched Target (1 MT), two base mismatched Target (2 MT), non-complementary DNA (NC), and No Target (No Target). And selectively characterizing the electrochemical DNA biosensor prepared by the invention, wherein:
the target DNA is single-stranded DNA, and the sequence is as follows:
5'-TCATCACACTGGAAGACTC-3'。
the single-base mismatched target DNA is single-stranded DNA, and the sequence is as follows:
5'-TCATCACACTGGAAGAATC-3'。
the two-base mismatched target DNA is single-stranded DNA, and the sequence is as follows:
5'-TCATCACACTGGAAGGATC-3'。
the non-complementary target DNA is single-stranded DNA, and the sequence is as follows:
5'-GACGTCAGACTTCCTGCGA-3'。
as can be seen from fig. 5, NC differs less from No Target (No Target) for different DNA sequences at the same concentration as the Target, the electrochemical signal is very low, NC differs less from No Target for different DNA sequences at the same concentration as the Target, the electrochemical signal is very low, and the 1MT and 2MT peak current values are 61.6% and 33.7% of the Target peak current value, respectively. The result shows that the nucleic acid biosensor constructed by the strategy has good selectivity and base mutation analysis distinguishing capability on target DNA, and has great application potential in the aspect of detecting single nucleotide polymorphism.
Example 11: practicality detection of biosensors
In order to further verify the practicability of the biosensor prepared by the invention, target DNA in complex biological samples (2% human serum) was detected. 1-8 are respectively: (1) 40nM, (2) 10nM, (3) 4nM, (4) 200pM, (5) 60pM, (6) 20pM, (7) 100fM, (8) 0. Comparing the results obtained in the buffer solution and diluted serum, respectively, it can be seen from FIG. 6 that the electrochemical response intensity of the target DNA added to the diluted serum increases simultaneously as the concentration of the target DNA added increases gradually from 0 to 40nM, indicating that the electrochemical response of the target DNA added to the diluted serum is substantially equivalent to that in the buffer solution. The sensor system prepared by the strategy of the invention has better detection capability in complex biological samples.

Claims (4)

  1. The p53 gene detection probe is characterized by comprising porous hollow magnetic nano-particles, electrochemical redox active probes encapsulated inside the porous hollow magnetic nano-particles, a cationic polymer functional layer coated on the surfaces of the porous hollow magnetic nano-particles and capture DNA adsorbed on the surfaces of the functional layer, wherein the capture DNA is a partial complementary sequence of p53 genes, and the sequence is 5'-TCTTCCAGTGTGATG-3';
    the porous hollow magnetic nanoparticle is selected from Fe 3 O 4 、Fe(O)、MnFe 2 O 4 、CoFe 2 O 4 、NiFe 2 O 4 Any one of them;
    the saidThe electrochemical redox active probe is [ Fe (CN) 6 ] 3- The cationic polymer is polydiallyl dimethyl ammonium chloride.
  2. 2. The method for preparing a p53 gene detection probe according to claim 1, comprising the steps of:
    adding the dried porous hollow magnetic nano particles into a cationic polymer solution, and carrying out ultrasonic treatment to obtain a uniform suspension;
    performing magnetic separation on the obtained suspension, and collecting a magnetic separation product to obtain porous hollow magnetic nano particles coated with cationic polymer;
    mixing porous hollow magnetic nano particles coated with cationic polymer with electrochemical redox active probe at 22-28 ℃ for 6-8h at 100-120r, adding capture DNA in Tris-HCl buffer system, and mixing at 22-28 ℃ for 100-120r for 6-8h to obtain magnetic composite material;
    and performing magnetic separation, washing and drying on the obtained magnetic composite material to obtain the p53 gene detection probe.
  3. 3. A biosensor for p53 gene detection, comprising the p53 gene detection probe according to claim 1.
  4. 4. The biosensor of claim 3, further comprising a modified electrode consisting of a 12-thiol modified gold electrode and methylene blue enriched at the 12-thiol surface.
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CN105758918A (en) * 2016-04-08 2016-07-13 青岛科技大学 Preparation and application method of electrochemical reduction graphene oxide and nanogold modified electrode based DNA sensor
CN107656059A (en) * 2017-09-26 2018-02-02 中南大学 A kind of Fluorescent detector for p53 albumen and its preparation method and application

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CN105758918A (en) * 2016-04-08 2016-07-13 青岛科技大学 Preparation and application method of electrochemical reduction graphene oxide and nanogold modified electrode based DNA sensor
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