CN113960142A - Preparation method of palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor - Google Patents
Preparation method of palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor Download PDFInfo
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Abstract
The invention provides a preparation method of a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor. The invention designs a preparation method of a palindromic nucleic acid nanosheet ultra-sensitive electrochemical biosensor based on a three-dimensional self-assembled DNA nanostructure triggered by a palindromic probe and combining the high fidelity of thermostable ligase and the inherent advantages of electrochemical measurement.
Description
Technical Field
The invention belongs to the technical field of nano materials, and particularly relates to a preparation method of a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor.
Background
The p53 gene is one of important tumor suppressor genes, which can mediate cell proliferation and apoptosis by encoding and expressing p53 protein, and plays a key role in preventing tumorigenesis. Base mutations in the p53 gene are considered to be common genetic alterations in human cancers. In addition to the loss of tumor suppressor activity, the mutated p53 gene also exhibits novel oncogenic activity to promote tumor development, progression, metastasis and drug resistance. Mutations in this gene in cancer patients have become clinically valuable biomarkers for early diagnosis, prognosis, risk of relapse assessment and potential therapeutic targets. Therefore, highly sensitive and reliable detection of the mutant p53 gene is crucial for cancer screening, prevention and treatment. Currently, various techniques for generating Amplification signals for disease-related gene detection, such as Polymerase Chain Reaction (PCR), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), Nicking Endonuclease Signal Amplification (NESA), and the like, have been developed. Despite significant advances in these technologies, these strategies have their own drawbacks that have hindered the transition from in vitro to clinical diagnostics and other applications. For example, PCR has been recognized as a mature nucleotide index amplification technique and is widely used to detect genes by amplifying molecular hybridization events. However, it requires special equipment to precisely control the temperature cycling and faces the risk of cross-contamination associated with the target amplification process. Similarly, RCA strongly depends on relatively long padlocks, whereas SDA is generally limited by assay capabilities at picomolar to sub-picomolar levels, and does not support direct detection of low-abundance target species without sequence pre-amplification. At the same time, enzymatic amplification-based assays suffer from the disadvantages of easy cross-contamination, inherent vulnerability to changes in environmental conditions (e.g., pH and temperature), false positive results from non-specific catalytic amplification, and complex procedures. In addition, a target gene with a single base mismatch can be hardly distinguished from a wild gene. In order to circumvent these limitations and improve the ability to recognize single base polymorphisms, it is urgently required to develop a simple enzyme-free signal amplification technique for screening for point mutations present in a target gene.
Because of their inherent advantages, such as simple operation, low cost, high efficiency of isothermal amplification and no need for enzyme-mediated Hybridization Chain Reaction (HCR), has become a versatile tool for signal amplification in various bioassays, and its constituent elements include: the priming probe and two hairpin DNAs (H1 and H2) which hybridize complementary and have sticky ends. H1 and H2 can exist stably if no priming sequence exists. Once the initiation probe exists, the secondary structure of the hairpin H1 is opened by the initiation probe, the stem end released by H1 opens the secondary structure of the hairpin H2, the stem end released by H2 has the same sequence as the initiation probe, and the secondary structure of H1 is opened, and H1 and H2 are opened mutually in such a reciprocating cycle, and finally a double-chain linear alternating copolymer containing a gap is formed. However, the detection sensitivity of linear HCR is not sufficient to report disease-related biomarkers in clinical applications. Thus, non-linear HCRs, such as branched HCRs, dendritic HCRs and stacked HCRs, are of increasing interest due to their second and exponential signal amplification. However, nonlinear HCRs typically require more different DNA hairpins to drive nanostructure growth, which makes probe design more difficult, increases detection costs and requires longer reaction times to be maximal. Furthermore, non-linear HCRs may involve more complex assembly processes, such as a clean-up step to remove residual building blocks. Therefore, it is highly desirable to assemble high order periodic and compact DNA nanostructures through programmable interactions (e.g., interactions between palindromic sticky ends) using a very small number of unique DNA strands as building blocks.
For nucleic acids containing palindromic fragments, hybridization between strands of the same type can occur through palindromic-based interactions without the aid of any helper probes. For example, as palindromic fragments, we can hybridize 5'-CCGTACGG-3' to 3'-GGCATGCC-5' in the 3'→ 5' orientation, producing double-stranded (ds) duplexes. By designing the palindromic segments as building blocks, the assembly system can be greatly simplified. Palindromic DNA strands are suitable for the controlled construction of well-defined DNA nanostructures of different geometries, including one-dimensional DNA nanotubes, two-dimensional DNA arrays, and three-dimensional supramolecular polyhedra, as well as configurable DNA networks and DNA microparticles. Furthermore, palindromic-mediated DNA nanotechnology allows for the direct self-assembly of predictable, programmable DNA structures onto the surface of solid substrates without the need for pre-construction processes in homogenous solutions. Also, the palindromic-based assay system or DNA component can improve signal sensitivity and payload capacity, and therefore has particular promise for biomedical bioanalytical analysis and drug delivery applications, such as detection of disease-related biomarkers, intracellular imaging of important genetic regulators in various pathological and physiological processes, real-time monitoring of in vivo tumor evolution, and targeted cancer therapy as a nuclease-resistant drug delivery nanocarrier.
Based on the reasons, the preparation method of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor is designed based on the three-dimensional self-assembled DNA nanostructure triggered by the palindromic probe and combining the high fidelity of the thermostable ligase and the inherent advantages of electrochemical measurement (such as high sensitivity, simple operation, rapid response and good compatibility with other signal transduction technologies). The ultrasensitive electrochemical detection biosensor prepared by the preparation method can perform double-signal amplification by combining surface functionalization based on connection with palindrome-mediated HCR, so that gene point mutation can be distinguished in an ultrasensitive manner. On the surface of the electrochemical biosensor, target-mediated immobilization of Trigger Probes (TP) activates self-assembly of three-dimensional DNA nanoplates (3D SDN) only from two hairpin DNA building blocks. The self-assembled 3D SDN material has a high loading capacity, can accommodate a large amount of the electrochemical indicator Methylene Blue (MB) and produces a strong electrochemical signal. By using the 3D SDN-based electrochemical biosensor, target DNA with lower concentration can be detected, the linear response range is wide, and mutant genes with single-point mutation can be easily distinguished from wild-type genes even under complex biological environments (such as fetal calf serum and cell homogenate). The palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor constructed based on the three-dimensional self-assembled DNA nanostructure initiated by the palindromic probe is expected to provide a potential platform for point mutation analysis required by early clinical diagnosis and medical research, and also provides a substitute tool for screening genetic polymorphism to implement cancer diagnosis, prognosis and accurate treatment.
Disclosure of Invention
The invention aims to provide a preparation method of a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor based on an assembly technology for preparing 3D SDNA three-dimensional nanosheets by using two types of palindromic DNA hairpin probes.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor comprises the following steps:
(1) polishing the bare gold electrode with alumina polishing powder until the surface is smooth, ultrasonically washing the bare gold electrode in deionized water, immersing the washed gold electrode in newly-configured Piranha solution to eliminate organic pollution, cleaning the gold electrode with deionized water, and airing the gold electrode at room temperature to obtain a pretreated gold electrode; dripping 10 mu L of 1 mu M thiolated capture probe on the surface of the pretreated gold electrode to completely cover a working interface of the gold electrode, incubating for 12 hours at room temperature in a water saturated atmosphere, then washing the gold electrode with ultrapure water, and then sealing non-specific adsorption sites on the surface of the electrode by adopting 6-mercapto-1-hexanol to obtain a probe-modified working electrode;
(2) dripping 10 mu L of target probe on the surface of the working electrode modified by the probe obtained in the step (1), incubating at room temperature for 1 h, and then putting the working electrode into 0.1M PBS buffer solution with pH7.4, stirring and washing; continuously dropwise adding 10 mu L of 1 mu M trigger probe on the surface of the washed electrode, incubating for 1 h at room temperature, and then placing the working electrode in 0.1M PBS buffer solution with pH7.4, stirring and washing; dripping 10 mu L of 10U Taq DNA Ligase on the surface of the washed working electrode, and reacting for 2 h at room temperature; after the reaction is finished, the working electrode is soaked in hot water at 95 ℃ for 5 min to release the target DNA thermally, then the working electrode is transferred to another tube of water with the same temperature, and then the working electrode is cooled to room temperature gradually; the process of target DNA hybridization-connection-heat release is carried out three times in total;
(3) uniformly mixing 10 mu L of 1 mu M palindromic probe 1 and an equivalent palindromic probe 2, dripping the mixture on the surface of the working electrode treated in the step (2), and then incubating for 2 h at room temperature; after the incubation is finished, washing the working electrode by using 0.1M PBS buffer solution with pH7.4, dripping methylene blue on the surface of the working electrode, and incubating for 40 min at room temperature to obtain the working electrode to be detected;
(4) washing a working electrode to be detected by using 0.1M PBS buffer solution with the pH value of 7.4, then forming a three-electrode system by using a saturated calomel electrode as a reference electrode and a platinum wire as an auxiliary electrode, placing the three-electrode system in a working solution, performing cyclic voltammetry scanning on an electrochemical workstation, and collecting generated electrochemical signals.
In the preparation method, the nucleotide sequence of the palindromic probe 1 is as follows: 5'-CCGTACGGTTTCGTCGTGCAGCAGCAGCAGCAGCAACGGCTTGCTGCTGCTGCTGCTGC-3' the flow of the air in the air conditioner,
the palindromic probe 2 nucleotide sequence is: 5'-TGCTGCTGCTGCTGCTGCACGACGGCAGCAGCAGCAGCAGCAAGCCGTTTTGATCGATC-3' are provided.
In the preparation method, the nucleotide sequence of the target probe is as follows: 5'-GGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGG-3', 5'-GGAACAGCTTTGAGGTGCtTGTTTGTGCCTGTCCTGGG-3', 5'-GGAACAGCTTTGAGGTGCcTGTTTGTGCCTGTCCTGGG-3',
5’-GGAACAGCTTTGAGGTGCaTGTTTGTGCCTGTCCTGGG-3’、
5’-GGAACAGCTTTtAGGTGCaTGTTTGTGCCTGTCCTGGG-3’、
5’-GGAACAGCTTTtAGGTGCaTGTTTGTtCCTGTCCTGGG-3’、
5'-GGAACAGCTTTtAGtTGCaTGTTTGTtCCTGTCCTGGG-3' at any one of the time points,
the nucleotide sequence of the thiolated capture probe is as follows: 5 '-P-CGCACCTCAAAGCTGTTCCTTTTTTTTTT-SH-3',
the trigger probe nucleotide sequence is as follows: 5'-TGCTGCTGCTGCTGCTGCACGACGCCCAGGACAGGCACAAACA-3' are provided.
In the preparation method, the nucleotide sequence of the target probe is as follows: 5'-TTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCC-3' at the time of the start of the operation,
the nucleotide sequence of the thiolated capture probe is as follows: 5 '-P-CCAGCTCCAACTACCACAATTTTTTTTTT-SH-3',
the trigger probe nucleotide sequence is as follows: 5'-TGCTGCTGCTGCTGCTGCACGACGGGCACTCTTGCCTACGCCA-3' are provided.
A palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor prepared by the preparation method.
By changing the sequences of the capture probe, the target probe and the trigger probe in the preparation method, a general electrochemical biosensor for specifically detecting the point mutation of the gene corresponding to the target probe can be constructed.
The invention principle of the invention is as follows:
first, a Capture Probe (CP) with a 5' -PO3 moiety was immobilized on the polished gold electrode surface by Au-S bond interaction, and the remaining bare area of the electrode surface was blocked with 6-mercapto-1-hexanol (MCH). Subsequently, the gold electrode sensing surface was incubated in a mixture of target probe, Trigger Probe (TP) and thermostable ligase, and then the annealing treatment step was repeated and washed by hot water soaking. To screen for point mutations, CP and TP are designed to be perfectly complementary to the target probe and hybridization between them can form a typical sandwich triplex, with the nick located near the point mutation that may be carcinogenic. Thus, only when the target probe is present and there are no mismatched base pairs in the triplex, the TP can be ligated to the CP and remain on the electrode surface during the washing step. Other nucleic acid probes, including hybridized target strands, are heat washed off the electrode surface. When exposed to two metastable hairpin DNA probes (HP 1 and HP 2), the surface-confined TP will initiate a palindromic-mediated HCR (p-HCR) process, thereby enabling self-assembly of three-dimensional DNA nanoplates (3D SDNA). It is well known that Methylene Blue (MB) is a redox active indicator that can accumulate on the electrode surface by interacting with guanine or inserting a double strand, and that surface-immobilized DNA material can mediate the transport of charge MB. Thus, enhanced electrochemical signals can be detected after treatment of the 3D SDNA assembled electrode with the MB solution.
Since the interaction between HP1 and HP2 is locked by their stem portions, the short palindromic fragments do not promote the formation of stable hybrids of HP1/HP1 or HP2/HP2, and thus HP1 and HP2, respectively, are stable in solution. Upon encountering the TP probe, HP1 is opened by hybridization, releasing a 24-base fragment complementary to HP 2. Thus, the metastable HP2 opens, opening the next HP1, breaking the metastable state of the two hairpin probes, activating the HCR reaction. HP1 and HP2 were arranged alternately in the horizontal direction to form a long continuous ds DNA line (L-HCR product) in which palindromic fragments were arranged in a regular repeating pattern. Identical palindromic ends arranged on adjacent L-HCR products can interact, resulting in lateral cross-linking between different L-HCRs in different directions and producing an interwoven 3D (rather than 2D) SDNA structured surface on the electrode. The entire hybridization process is called palindromic-mediated hybrid strand reaction (p-HCR), in which only two hairpin DNA building blocks are involved.
The invention has the advantages that:
the invention designs a preparation method of a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor based on a three-dimensional self-assembled DNA nanostructure triggered by a palindromic probe, and the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor prepared by the method has the following advantages:
(1) by using a cohesive end-mediated three-dimensional nucleic acid nano-assembly technology, the 3D SDNA nano-sheet can be self-assembled only by two types of hairpin DNA short sequences.
(2) The electrochemical biosensor constructed by the palindromic nucleic acid nanosheet has good detection performance. Firstly, the electrochemical sensor uses MB molecules to embed into a three-dimensional self-assembly DNA nano-structure triggered by a palindromic probe for signal output, and the signal output is lower than that of a traditional electric signal conduction method depending on the configuration change of a nucleic acid probe; secondly, the trigger probe can trigger two palindromic hairpins to be alternately opened and generate a palindromic-mediated hybrid chain reaction, so that a 3D SDNA nano structure is finally formed, and the phenomenon indicates that one target molecule can introduce a plurality of signal response molecules, thereby improving the detection sensitivity of the electrochemical biosensor.
(3) The electrochemical biosensor constructed by the palindromic nucleic acid nanosheets can accurately identify point mutation of a target gene. The electrochemical biosensor has the capacity of distinguishing single nucleotide point mutation by utilizing the high fidelity characteristic of Taq DNA Ligase.
(4) The electrochemical biosensor constructed by the palindromic nucleic acid nanosheets can maintain excellent detection performance and selectivity in complex biological environments (such as fetal calf serum and cell homogenate).
Drawings
FIG. 1: a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor construction flow chart based on a palindromic probe-triggered three-dimensional self-assembled DNA nanostructure in example 1.
FIG. 2: schematic representation of the formation of three-dimensional DNA palindromic nucleic acid nanoplatelets under surface-confined TP triggering in example 1.
FIG. 3: a palindromic nucleic acid nanosheet material characterization map in the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor in example 1. Wherein 3A is polyacrylamide gel electrophoresis analysis of palindromic nucleic acid nanosheets: lane M represents a DNA standard band; lane 1 is palindromic probe 1 (HP 1); lane 2 is palindromic probe 2 (HP 2); lane 3 is a mixture of palindromic probe 1 and palindromic probe 2; lane 4 is a mixture of palindromic probe 1 and palindromic probe 2, to which a Trigger Probe (TP) related to the p53 gene was added. And 3B is an atomic force characterization image of a final three-dimensional self-assembled DNA nano structure, namely a palindromic nucleic acid nano sheet.
FIG. 4: the detection performance of the palindromic acid nanosheet ultrasensitive electrochemical biosensor in example 2 is analyzed, and the single nucleotide point mutation discriminative power of the palindromic acid nanosheet ultrasensitive electrochemical biosensor in example 3 is analyzed. Wherein 4A is feasibility analysis data of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor in example 2, the black dotted line is a detection signal of the electrochemical biosensor in the absence of the target probe p53 DNA, the red dotted line is a detection signal of the electrochemical sensor in the presence of the target probe p53 DNA, and the red solid line is a background signal. And 4B is the detection signal of the electrochemical biosensor of the target probe p53 DNA with different concentrations in example 2. 4C is the linear relation between the detection signal of the electrochemical biosensor and the concentration of the target probe p53 DNA in example 2. 4D is the discrimination ability of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor in example 3 for detecting single nucleotide point mutations occurring in the p53 gene, wherein red is wild-type target probe p53 DNA, the background-subtracted detection signal thereof is defined as 100%, green, blue and cyan are single base mutation probes (green is T base mutation, blue is C base mutation, and cyan is a base mutation), purple is a dibasic mutation probe, yellow is a three base mutation probe, and curry is a four base mutation probe.
FIG. 5: in example 4 and example 5, the detection performance of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor in a complex biological environment is analyzed. Wherein 5A is the detection performance analysis of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor in FBS (fetal bovine serum), a blank column is the concentration of a target probe which is actually added, and an oblique line column is the concentration of the target probe detected by the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor. And 5B, analyzing the detection performance of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor in cell homogenate, wherein a blank column is the concentration of a target probe actually added, and an oblique line column is the concentration of the target probe detected by the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor.
FIG. 6: example 6 evaluation of the versatility of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor. Wherein 6A is a schematic diagram of a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor using a three-dimensional self-assembled DNA nanostructure initiated by a palindromic probe for KRAS gene. And 6B is feasibility analysis data of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor for KRAS gene detection. The black dotted line is the detection signal of the electrochemical biosensor in the absence of the target probe, and the red dotted line is the detection signal of the electrochemical sensor in the presence of the target probe.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.
Example 1
The construction flow chart of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor based on the three-dimensional self-assembled DNA nanostructure triggered by the palindromic probe is shown in FIG. 1; a schematic diagram of the formation of 3D SDNA three-dimensional palindromic nucleic acid nanoplates under surface-constrained TP triggering is shown in figure 2.
A preparation method of a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor comprises the following steps:
(1) polishing bare gold electrode with diameter of 2 mm with 0.05 μm aluminum oxide polishing powder for 5 min to make its surface smooth, ultrasonic washing the polished gold electrode in deionized water for 5 min, and placing the washed gold electrode in newly-configured Piranha solution (H in Piranha solution)2SO4And H2O2The volume ratio of (1) to (3) for 10 min to eliminate organic pollution, then using deionized water to clean the electrode, and drying at room temperature to obtain a pretreated gold electrode; and dripping 10 mu L of 1 mu M thiolated Capture Probe (CP) on the surface of the pretreated gold electrode to completely cover the working interface of the gold electrode, incubating the gold electrode dripped with the CP probe in a water-saturated atmosphere at room temperature for 12 h in order to ensure that the CP probe is fully combined with the surface of the gold electrode and prevent the evaporation and drying of the water on the interface, washing the gold electrode by using ultrapure water, dripping 20 mu L of 1 mM 6-mercapto-1-hexanol (MCH) on the surface of the electrode, incubating for 10 min to seal the non-specific adsorption sites on the surface of the electrode, and obtaining the working electrode modified by the probe.
Wherein the CP probe nucleotide sequence is: 5 '-P-CGCACCTCAAAGCTGTTCCTTTTTTTTTT-SH-3'.
(2) Dripping 10 mu L of target probe p53 DNA onto the surface of the probe-modified working electrode obtained in the step (1), incubating at room temperature for 1 h, and then putting the working electrode in 0.1M PBS buffer (pH = 7.4) for stirring and washing; continuously dripping 10 mu L of 1 mu M Trigger Probe (TP) on the surface of the washed working electrode, incubating at room temperature for 1 h, and then continuously putting the working electrode into 0.1M PBS buffer (pH = 7.4) for stirring and washing; dripping 10 mu L of Taq DNA Ligase (10U) on the surface of the washed working electrode, and reacting for 2 h at room temperature; after the reaction is finished, the working electrode is soaked in hot water (95 ℃) for 5 min to release the target DNA thermally, then the working electrode is transferred to another tube of water with the same temperature, and then the working electrode is cooled to room temperature gradually; the target DNA hybridization-ligation-heat release process was performed three times in total.
Wherein the target probe p53 DNA nucleotide sequence is:
5’-GGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGG-3’。
the TP probe nucleotide sequence is as follows:
5’-TGCTGCTGCTGCTGCTGCACGACGCCCAGGACAGGCACAAACA-3’。
(3) uniformly mixing 10 mu L of 1 mu M palindromic probe 1 (HP 1) and an equivalent concentration palindromic probe 2 (HP 2), dropwise adding the mixture on the surface of the working electrode treated in the step (2), and then incubating for 2 h at room temperature to perform p-HCR-based three-dimensional DNA nano-material self-assembly; in order to remove the free nucleic acid probe on the electrode surface, the working electrode was washed with 0.1M PBS buffer (pH = 7.4), and then 10 μ L of 1 mM Methylene Blue (MB) was added dropwise to the surface of the working electrode and incubated at room temperature for 40 min, to obtain the working electrode to be tested.
(4) The working electrode to be tested was washed with 0.1M PBS buffer (pH = 7.4) and composed of a conventional three-electrode system with a platinum wire as auxiliary electrode and a saturated calomel electrode as reference electrode, and the resulting electrochemical signals were collected using an electrochemical workstation (all electrochemical measurements were performed on CHI660D electrochemical workstation; differential pulse voltammetry was performed in the range of-0.4V to 0.1V, pulse amplitude of 50 mV, pulse width of 1.1 s, pulse period of 0.5 s; 1 mM Fe (CN) containing 0.5M KCl was used)6 3-/4-The solution was used as a working solution, and electrochemical impedance spectroscopy measurements were performed in a frequency range of 0.1 Hz to 10 kHz while cyclic voltammetry measurements were performed from-0.2V to 0.6V at a scan rate of 0.1 mV/s).
Wherein the HP1 nucleotide sequence is: 5'-CCGTACGGTTTCGTCGTGCAGCAGCAGCAGCAGCAACGGCTTGCTGCTGCTGCTGCTGC-3' are provided.
The HP2 nucleotide sequence is: 5'-TGCTGCTGCTGCTGCTGCACGACGGCAGCAGCAGCAGCAGCAAGCCGTTTTGATCGATC-3' are provided.
FIG. 3A is a polyacrylamide gel electrophoresis analysis of palindromic nucleic acid nanoplatelets, wherein lane M represents a DNA standard band; lane 1 is palindromic probe 1 (HP 1); lane 2 is palindromic probe 2 (HP 2); lane 3 is a mixture of palindromic probe 1 and palindromic probe 2; lane 4 is a mixture of palindromic probe 1 and palindromic probe 2, to which a Trigger Probe (TP) related to the p53 gene was added. As can be seen in FIG. 3A, in the absence of the target sequence, both palindromic probe 1 (HP 1) and palindromic probe 2 (HP 2) are in a relatively stable monomeric state (lanes 1-3); when the trigger probe exists, the gel electrophoresis migration rate of the DNA is obviously changed, and an assembly product with different strips is generated, which indicates that the trigger probe can trigger the assembly of the three-dimensional DNA nano material.
Fig. 3B is an atomic force characterization image of the final three-dimensional self-assembled DNA nanostructure, palindromic nucleic acid nanosheets. And (3) performing structural morphology characterization on the finally formed palindromic acid nanosheet structure by using an atomic force microscope, wherein the structure is shown in fig. 3B, and the finally formed palindromic acid nanosheet structure can be seen as an oval, and has a width and a height, which proves that a three-dimensional nanosheet structure can be formed by two palindromic sequences of HP1 and HP 2.
Example 2
Only the concentration of the target probe p53 DNA is changed, a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor is constructed according to the method in example 1, and an electrochemical workstation is used for detecting the electrochemical signal of the constructed palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor.
Fig. 4A is a feasibility analysis experiment of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor. The black dotted line is the detection signal of the electrochemical biosensor in the absence of the target probe p53 DNA, the red dotted line is the detection signal of the electrochemical sensor in the presence of the target probe p53 DNA, and the red solid line is the background signal. As can be seen from FIG. 4A, the electrochemical biosensor generates a distinct electrochemical signal in the presence of the target probe p53 DNA, which is far distinguished from the background signal (black dashed line) and the electrochemical signal in the absence of the target probe.
FIG. 4B shows the detection signals of the electrochemical biosensor under the conditions of different concentrations of target probe p53 DNA, and it can be seen that the electrochemical signals gradually increase with the increase of the concentration of target probe p53 DNA in the range of 10 fM to 10 nM, indicating that more target species can indeed self-assemble into more three-dimensional DNA nano-material structures and can adsorb more redox Molecules (MB) on the electrode surface. In addition, a good linear relationship was achieved between the peak current and the logarithm of the target probe concentration (fig. 4C).
Example 3
Only the sequence of the target probe p53 DNA is changed, a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor is constructed by referring to the method in example 1, and an electrochemical workstation is used for detecting the electrochemical signal of the constructed palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor.
The DNA strands used were as follows:
the nucleotide sequence of the T base mutation probe is as follows: 5'-GGAACAGCTTTGAGGTGCtTGTTTGTGCCTGTCCTGGG-3', respectively;
the nucleotide sequence of the C base mutation probe is as follows: 5'-GGAACAGCTTTGAGGTGCcTGTTTGTGCCTGTCCTGGG-3', respectively;
the nucleotide sequence of the A base mutation probe is as follows: 5'-GGAACAGCTTTGAGGTGCaTGTTTGTGCCTGTCCTGGG-3', respectively;
the nucleotide sequence of the two-base mutation probe is as follows: 5'-GGAACAGCTTTtAGGTGCaTGTTTGTGCCTGTCCTGGG-3', respectively;
the nucleotide sequence of the three-base mutation probe is as follows: 5'-GGAACAGCTTTtAGGTGCaTGTTTGTtCCTGTCCTGGG-3', respectively;
the four-base mutation probe nucleotide sequence is as follows: 5'-GGAACAGCTTTtAGtTGCaTGTTTGTtCCTGTCCTGGG-3' are provided.
FIG. 4D is the data of the discrimination ability of the corresponding palindromic acid nanosheet ultrasensitive electrochemical biosensor on single nucleotide point mutation. Wherein red is a wild-type target probe p53 DNA (the target probe p53 DNA sequence is the same as in example 1), the detection signal with background subtracted is defined as 100%, green, blue and cyan are single base mutations (green is a T base mutation, blue is a C base mutation, and cyan is an A base mutation), purple is a two base mutation, yellow is a three base mutation, and curry is a four base mutation.
The formula for calculating the relative current response (Rc) is as follows: rc (Ct-Cb)/(Cw-Cb). times.100%, where Cw, Ct and Cb correspond to the peak currents of the wild-type target probe p53 DNA, mutant target probe and blank sample, respectively. As can be seen from the figure, the electrochemical signal induced by the non-mutated target probe p53 DNA is significantly higher than that induced by the rest of the mutated target probes. Specifically, defining the signal on the wild-type target probe p53 DNA as 100%, all other mutant target probes caused a relative current response of less than 10%. The series of data indicate that the constructed three-dimensional self-assembly DNA nanostructure triggered by the palindromic probe can be used for detecting point mutation of p53 gene and has the capacity of distinguishing single nucleotide point mutation.
Example 4
Firstly, target probe p53 DNA sequences with different concentrations are added into 2 mL10% (v/v) fetal calf serum and fully mixed (the final target probe concentrations are 1 pM, 10 pM, 100 pM and 1000 pM respectively), then a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor is constructed according to the method of example 1, and electrochemical signals are detected by using an electrochemical workstation, only 10 μ L of the target probe p53 DNA sequence is replaced by the mixture of the fetal calf serum and the target probe p53 DNA sequence.
Fig. 5A is a corresponding analysis of the detection performance of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor in FBS, where a blank column is the target probe concentration actually added, and an oblique column is the target probe concentration detected by the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor. As can be seen from the figure, the concentration of the p53 target probe detected by the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor is basically consistent with the concentration of the artificially and actually added target probe, which indicates that the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor can be applied to detection in a complex biological environment.
Example 5
Culturing L02 liver cell in 5 mL RPMI-1640 medium containing 10% fetal calf serum and 100 IU/mL penicillin-streptomycin double antibody at 37 deg.C and 5% CO2And 95% relative humidity of carbon dioxideCulturing in a cell culture box for 24 h; the L02 liver cells (1.0X 10)7Respectively) centrifuging at 25 deg.C and 3000 rpm for 3 min to remove supernatant, then resuspending the precipitate in 2 mL RIPA lysis buffer (purchased from Shanghai Biotech engineering Co., Ltd.), and incubating at room temperature for 10 min to obtain L02 hepatocyte homogenate; immediately adding p53 DNA target probe sequences with different concentrations into 2 mL of cell homogenate and mixing thoroughly (the final concentrations of the target probes are 1 pM, 10 pM, 100 pM and 1000 pM respectively), and then constructing a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor with reference to the method of example 1 and detecting electrochemical signals using an electrochemical workstation, except that 10. mu.L of the target probe p53 DNA sequence was replaced by the aforementioned mixture of cell homogenate and the target probe p53 DNA sequence.
FIG. 5B is a performance analysis of the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor in cell homogenate. The blank column is a p53 target probe which is actually added, and the diagonal column is the concentration of the target probe detected by the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor. As can be seen from the figure, the concentration of the p53 target probe detected by the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor is basically consistent with the concentration of the artificially and actually added target probe, which indicates that the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor can be applied to detection in a complex biological environment.
Example 6
Only the sequences of the capture probe, the target probe and the trigger probe are changed, a palindromic nucleic acid nanosheet ultra-sensitive electrochemical biosensor is constructed according to the method in example 1, and an electrochemical workstation is used for detecting electrochemical signals of the constructed ultra-sensitive electrochemical sensor.
The DNA strands used were as follows:
the KRAS capture probe nucleotide sequence is: 5' -P ' -CCAGCTCCAACTACCACAATTTTTTTTTT-SH-3 ';
the Target KRAS Target probe nucleotide sequence is as follows: 5'-TTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCC-3', respectively;
the KRAS trigger probe nucleotide sequence is as follows: 5'-TGCTGCTGCTGCTGCTGCACGACGGGCACTCTTGCCTACGCCA-3' are provided.
FIG. 6A is a schematic diagram of construction of a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor using a palindromic probe-triggered three-dimensional self-assembled DNA nanostructure. Fig. 6B shows the detection feasibility of the palindromic acid nanosheet ultrasensitive bioelectrochemical sensor on KRAS gene, and it can be seen from the figure that the electrochemical biosensor shows ideal detection performance in the presence of Target KARS Target probe. Through the data analysis, the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor capable of detecting different target genes can be constructed only by changing the trigger probe and the capture probe, and the fact that the palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor provided by the invention has better universality and application potential is proved.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fuzhou university
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Claims (5)
1. A preparation method of a palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor is characterized by comprising the following steps: the method comprises the following steps:
1) polishing the bare gold electrode with alumina polishing powder until the surface is smooth, ultrasonically washing the bare gold electrode in deionized water, immersing the washed gold electrode in newly-configured Piranha solution to eliminate organic pollution, cleaning the gold electrode with deionized water, and airing at room temperature to obtain a pretreated gold electrode; dripping 10 mu L of 1 mu M thiolated capture probe on the surface of the pretreated gold electrode to completely cover a working interface of the gold electrode, incubating for 12 hours at room temperature in a water saturated atmosphere, washing the gold electrode with ultrapure water, and sealing non-specific adsorption sites on the surface of the electrode by adopting 6-mercapto-1-hexanol to obtain a probe-modified working electrode;
2) dripping 10 mu L of target probe on the surface of the working electrode modified by the probe obtained in the step (1), incubating at room temperature for 1 h, and then putting the working electrode into 0.1M PBS buffer solution with pH7.4, stirring and washing; continuously dropwise adding 10 mu L of 1 mu M trigger probe on the surface of the washed electrode, incubating for 1 h at room temperature, and then placing the working electrode in 0.1M PBS buffer solution with pH7.4, stirring and cleaning; dripping 10 mu L of 10U Taq DNA Ligase on the surface of the washed working electrode, and reacting for 2 h at room temperature; after the reaction is finished, the working electrode is soaked in hot water at 95 ℃ for 5 min to release the target DNA thermally, then the working electrode is transferred to another tube of water with the same temperature, and then the working electrode is cooled to room temperature gradually; the process of target DNA hybridization-connection-heat release is carried out three times in total;
3) uniformly mixing 10 mu L of 1 mu M palindromic probe 1 and an equivalent palindromic probe 2, dripping the mixture on the surface of the working electrode treated in the step (2), and then incubating for 2 h at room temperature; after the incubation is finished, washing the working electrode by using 0.1M PBS buffer solution with pH7.4, then dripping methylene blue on the surface of the working electrode, and incubating for 40 min at room temperature to obtain the working electrode to be detected;
4) washing a working electrode to be detected by using 0.1M PBS buffer solution with the pH value of 7.4, then forming a three-electrode system by using a saturated calomel electrode as a reference electrode and a platinum wire as an auxiliary electrode, placing the three-electrode system in a working solution, performing cyclic voltammetry scanning on an electrochemical workstation, and collecting generated electrochemical signals.
2. The method of claim 1, wherein:
the palindromic probe 1 has the nucleotide sequence as follows: 5'-CCGTACGGTTTCGTCGTGCAGCAGCAGCAGCAGCAACGGCTTGCTGCTGCTGCTGCTGC-3', respectively;
the palindromic probe 2 nucleotide sequence is: 5'-TGCTGCTGCTGCTGCTGCACGACGGCAGCAGCAGCAGCAGCAAGCCGTTTTGATCGATC-3' are provided.
3. The method of claim 1, wherein:
when the target probe nucleotide sequence is: 5'-GGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGG-3', 5'-GGAACAGCTTTGAGGTGCtTGTTTGTGCCTGTCCTGGG-3', 5'-GGAACAGCTTTGAGGTGCcTGTTTGTGCCTGTCCTGGG-3',
5’-GGAACAGCTTTGAGGTGCaTGTTTGTGCCTGTCCTGGG-3’、
5’-GGAACAGCTTTtAGGTGCaTGTTTGTGCCTGTCCTGGG-3’、
5’-GGAACAGCTTTtAGGTGCaTGTTTGTtCCTGTCCTGGG-3’、
5'-GGAACAGCTTTtAGtTGCaTGTTTGTtCCTGTCCTGGG-3' at any one of the time points,
the capture probe nucleotide sequence is: 5 '-P-CGCACCTCAAAGCTGTTCCTTTTTTTTTT-SH-3',
the trigger probe nucleotide sequence is as follows: 5'-TGCTGCTGCTGCTGCTGCACGACGCCCAGGACAGGCACAAACA-3' are provided.
4. The method of claim 1, wherein:
the target probe nucleotide sequence is as follows: 5'-TTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCC-3' at the time of the start of the operation,
the capture probe nucleotide sequence is: 5 '-P-CCAGCTCCAACTACCACAATTTTTTTTTT-SH-3',
the trigger probe nucleotide sequence is as follows: 5'-TGCTGCTGCTGCTGCTGCACGACGGGCACTCTTGCCTACGCCA-3' are provided.
5. A palindromic nucleic acid nanosheet ultrasensitive electrochemical biosensor prepared using the preparation method of claim 1.
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