CN111118117B

CN111118117B - DNA walker for relative movement between particles and application of DNA walker in biosensing

Info

Publication number: CN111118117B
Application number: CN201911318816.8A
Authority: CN
Inventors: 姜玮; 张宏; 徐晓文
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2019-12-19
Filing date: 2019-12-19
Publication date: 2021-11-02
Anticipated expiration: 2039-12-19
Also published as: CN111118117A

Abstract

The invention particularly relates to a DNA walker for relative movement between particles and application of biosensing of the DNA walker. The invention provides a DNA walker with relative movement among particles, which consists of WPs and TPs and is realized by inducing the WP to move relatively along the surrounding TP under the stimulation of a specific target. The WP continuously walks along the plurality of TPs one by one, so that the walking affinity is enhanced, and the walking freedom degree and the walking area are improved. In addition, the DNA walker has the rapid signal accumulation and anti-interference capability, and realizes the sensitive determination of the ZIKV-RNA in a complex matrix. The novel DNA walker has potential application value in sensitive detection of biomolecules and analysis of actual samples. Furthermore, the construction of a DNA machine based on phase separation modules can provide a powerful tool for regulating particle motion and studying interactions between surface immobilized molecules.

Description

DNA walker for relative movement between particles and application of DNA walker in biosensing

Technical Field

The invention belongs to the technical field of DNA walkers, and particularly relates to a DNA walker with relative movement among particles and application of the DNA walker in RNA nucleic acid fragment detection.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

DNA is considered to be one of the most promising molecular machine building materials due to its precise base pairing ability, sequence programmability, structural controllability, and ease of modification. DNA walker is a type of DNA molecular machine in which nucleic acids can move spontaneously and stepwise along predetermined trajectories. Are of particular interest due to their unique mechanical motion, high directivity, and inherent signal amplification capabilities. DNA walker has evolved from a one-dimensional (1-D) based trajectory to a two-dimensional (2-D) trajectory based on a plane of DNA folding or DNA modification, and even a three-dimensional (3D) trajectory based on spherical particles. Compared with 1-D and 2-D DNA walkers, the 3-D DNA walkers can walk more steps, because the 3-D DNA walkers have larger specific surface area to bear high-density DNA tracks, and the 3-D DNA walkers are endowed with higher operation persistence and better signal amplification capability.

The 3-D DNA walker constructed at present is generally divided into two types: immobilized DNA walker and episomal DNA walker. The immobilized DNA walker fixes one end of a walking chain to the surface of a particle, usually by covalent bonding of the walking chain to the surface of the particle or by bonding the walking chain to an auxiliary chain. Such 3-D DNA walkers typically modify the walking and orbital strands on the same spherical particle. The movement of the walking chain is limited to the surface of the particle and the limited length of the walking chain can only allow it to swing around the foothold, so the walking area of the 3-D DNA walker may be limited. The working principle of episomal DNA walker is that free one-or two-footed walking chains walk along the surface of the rail chain functionalized particles. During the running of the episomal DNA walker, the walking strand may be separated from the track to pause the step, and then stably reconnect to another DNA track. In general, limited walking areas or derailments may reduce walking speed and persistence, which further impairs the fast signal accumulation capability of 3-D DNA walker. The inventor considers that the above defects of the existing DNA walker are not beneficial to the continuous operation capability and detection sensitivity of the DNA walker in biological detection application, and limit the analysis capability of the DNA walker.

Disclosure of Invention

Against the background of the above-mentioned research, the inventors thought that it was necessary to develop a novel DNA walker, and the present invention provides a DNA walker of relative movement between particles, which can continue walking rapidly and has an improved degree of freedom of walking and an enhanced affinity to the track. The DNA walker consists of Walking Particles (WPs) and orbital particles (TPs). WPs was obtained by functionalizing blocked walking chains comprising DNAzyme sequences onto AuNPs. TPs are obtained by functionalizing fluorophore-tagged orbital strands comprising the substrate sequence onto AuNPs, and under the trigger of a stimulus, the walking strands on the WP hybridize synergistically with the orbital strands on the TP. The orbital strand is cleaved by DNAzyme with concomitant release of the fluorophore and recovery of fluorescence. The adjacent running strand on the WP then hybridizes to the next orbital strand, thereby inducing relative movement of the WP around the TP. After walking along the surface of one TP, WP looks for a new TP, continues to make relative motion and repeats the above process. The cooperative combination of the traveling chain and the track chain is stable, and WP is not easy to derail from TP. Compared with the DNA walker running in the particles, the designed DNA walker has the advantages that the walking freedom degree and the walking area are improved, the walking speed and the walking continuity are further improved, and the rapid accumulation of signals is finally promoted. The invention takes the pathogenic virus fragment ZIKV-RNA as a model target to verify the detection performance of the DNA walker, and proves that the DNA walker has good sensitivity and specificity.

Based on the research cost, the invention provides the following technical scheme:

the DNA walker performs relative movement among particles, and comprises walking particles and track particles, wherein the walking particles are composed of first gold nanoparticles and closed walking chains, the walking chains are single-stranded DNA and comprise DNAzyme sequences, one end of each DNAzyme sequence is connected with the gold nanoparticles, and the other end of each DNAzyme sequence is combined with the closed chains; the closed strand comprises a complementary sequence of a target to be detected;

the track particles are composed of second gold nanoparticles and a track chain marked by a fluorescent group, and the track chain is single-stranded DNA and comprises a sequence complementary with a walking chain;

the DNA walker further comprises a supplementary component for providing Mg²⁺。

Preferably, the walking chain comprises the 8-17E DNAzyme sequence.

Preferably, the particle size of the first gold nanoparticle and/or the second gold nanoparticle is 10-15 nm.

Preferably, the first gold nanoparticles and/or the second gold nanoparticles are HAuCl₄Is prepared by a citric acid reduction method.

The construction and operation principle of the DNA walker is shown in the attached figure 1. The DNA walker consists of two DNA functionalized AuNPs, the closed walking chain functionalized AuNPs are represented as WPs, and the orbital chain functionalized AuNPs are represented as TPs. Wherein, the walking chain comprises a sequence of 8-17E DNAzyme, the sequence is a single-stranded DNA fragment with high catalytic activity and structure recognition capability, and the closed chain comprises a complementary sequence of a target sequence. The track chain comprising DThe NA-RNA chimeric substrate sequence was labeled with the fluorescent dye carboxyfluorescein (FAM), which was initially quenched by proximity to the AuNPs. In the presence of the target sequence, the target sequence and the closed strand are hybridized to form a double-stranded product through strand displacement reaction, and the walking strand is released at the same time. Then, the walking strand on the WP is hybridized synergistically with the orbital strand. Using Mg²⁺As a cofactor, the orbital strand is cleaved by DNAzyme sequences, and the fluorophore FAM generates a fluorescent signal away from the AuNPs. The adjacent walking strand on the WP hybridizes with the next orbital strand of the TP, inducing relative movement of the WP around the TP, accompanied by stepwise cleavage of the orbital strand. After the WP finishes walking along the surface of one TP, it will look for a new TP, continue the relative motion and repeat the above process, releasing a large amount of fluorophore and producing signal accumulation. However, when the target sequence is not present, the DNAzyme sequence on the walking chain is blocked, its catalytic activity is inhibited, and thus DNA walker operation cannot be triggered to obtain a weak background signal.

In a second aspect of the present invention, there is provided a method for preparing the DNA walker with relative movement between particles in the first aspect, the method comprising the steps of:

mixing the walking chain and the closed chain, closing the walking chain in a heating annealing mode, reducing DNA disulfide bonds in the walking chain, mixing the walking chain with the first gold nanoparticles, adding NaCl in the stirring process, continuously stirring, performing a water washing step, and finally dispersing the modified first gold nanoparticles in a Tris-HCl buffer solution to obtain walking particles;

and mixing and stirring the reduced orbital chain and the second gold nanoparticles, and obtaining the orbital particles through the steps of adding salt, washing with water and dispersing in Tris-HCl buffer solution again.

Preferably, the ratio of the walking chain to the closed chain is 0.8-1.2: 4-6.

Preferably, the walking chain and the closed chain are mixed and heated to 88-92 ℃.

Preferably, the molar ratio of the blocked walking chains to the first gold nanoparticles is 140-160: 1.

Preferably, the molar ratio of the rail chain to the second gold nanoparticles is 190-210: 1.

In a third aspect of the invention, there is provided the use of the DNA walker for relative movement between particles described in the first aspect in the preparation of a nucleic acid biosensor.

Preferably, the nucleic acid biosensor is used for detecting RNA nucleic acid fragments, and comprises RNA sequences related to biomolecules, including RNA virus specific RNA fragments.

In a fourth aspect of the present invention, there is provided a nucleic acid biosensor comprising the DNA walker in relative motion between particles of the first aspect.

Preferably, the detection method of the nucleic acid biosensor is as follows: the sequence to be detected, the walking particles and MgCl₂Adding the mixed solution into a buffer solution to obtain a mixed solution, incubating for a period of time, adding the rail particles, reacting, and centrifuging to detect the fluorescence in the supernatant after reacting for a period of time.

Further preferably, the buffer solution is a Tris-HCl buffer solution with the pH value of 8-9 and containing NaCl.

Further preferably, the mixed solution is incubated at 35-39 ℃ for 1-2 hours.

Further preferably, the reaction time is 2 to 3 hours.

In a fifth aspect of the present invention, there is provided a nucleic acid detection kit comprising the DNA walker which moves relatively between particles as described in the first aspect.

Preferably, the detection kit further comprises a buffer solution.

Compared with the prior art, the beneficial effect of this disclosure is:

compared with the existing DNA walker, the invention provides a mode that the DNA walking chain and the track chain are respectively arranged on different nanoparticle carriers, and the walker is proved to be capable of actually passing through the relative movement among particles by comparing the DNA walker which moves relatively among the particles with the traditional DNA walker which moves relatively in the particles, so that the walking speed and the continuity of the walker are obviously improved; and under the same concentration, the signal accumulation degree is twice of that of the relative motion walker in the particles, thereby effectively improving the detection sensitivity. Besides, the walker has good detection specificity, and can be applied to the detection of samples in complex matrixes.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a schematic diagram of the principle of the inter-particle relative movement of DNA walker triggered by the target in the present invention.

FIG. 2 is a graph showing the characterization results of AuNPs synthesized in example 1;

wherein FIG. 2(A) is a UV-vis absorption spectrum;

fig. 2(B) is a TEM image.

FIG. 3 is a graph showing the modification and characterization results of WPs and TPs in example 1;

wherein FIG. 3(A) is a UV-vis absorption spectrum;

FIG. 3(B) is a graph showing WPs fluorescence spectra and an evaluation of the number of modified strands;

FIG. 3(C) is a graph showing the fluorescence spectrum of TPs and the evaluation of the number of modified orbital chains.

FIG. 4 is a graph showing the results of characterization of the DNA walker run for the relative movement between particles in example 1;

wherein 4(A) is the fluorescence emission spectrum of the DNA walker;

FIG. 4(B) is a non-denaturing PAGE analysis of DNA walker.

FIG. 5 is a graph showing the results of the kinetic study of the DNA walker with relative movement between particles in example 1;

wherein, FIG. 5(A) is the change of fluorescence of DNA walker in response to different target concentrations with reaction time;

FIG. 5(B) is the change in fluorescence of DNA walker in response to different target concentrations within the first 30 min;

FIG. 5(C) is the initial rate of DNA walker at different target concentrations. Error bars are the standard deviation of the results of three replicates.

FIG. 6 is a graph showing the results of comparing the performance of the DNA walker moving relatively between the particles with that of the DNA walker running in the particles in example 1;

wherein, FIG. 6(A) is the change of fluorescence intensity of the DNA walker moving relatively between the particles and the DNA walker moving within the particles with the reaction time;

FIG. 6(B) is fluorescence emission spectra of the DNA walker relatively moving between the particles and the DNA walker running inside the particles;

FIG. 6(C) is the reaction rates of the DNA walker moving relatively between the particles and the DNA walker running within the particles for the first 30 min. Error bars are the standard deviation of the results of three replicates.

FIG. 7 is a graph showing the characterization results of 5nm and 25nm AuNPs in example 1;

wherein FIG. 7(A) is a 5nm-AuNPs UV-vis absorption spectrum;

FIG. 7(B) is a 5nm-AuNPs TEM image;

FIG. 7(C) is a 5nm-AuNPs UV-vis absorption spectrum;

FIG. 7(D) is a 5nm-AuNPs TEM image.

FIG. 8 is a graph showing the results of fluorescence property evaluation of nanochains of different sizes in example 1;

wherein FIG. 8(A) is a graph showing the fluorescence spectrum of TP-Au5 and the evaluation of the number of modified track chains;

FIG. 8(B) is a graph showing the fluorescence spectrum of TP-Au25 and the evaluation of the number of modified track chains.

FIG. 9 is a Dynamic Light Scattering (DLS) characterization of TPs and WPs in example 1.

FIG. 10 is a graph showing the effect of changes in the size of TPs on the performance of DNA walker in relative movement between particles in example 1;

wherein, FIG. 10(A) is the change of fluorescence of DNA Walker of TPs of different sizes with the reaction time (same TPs concentration);

FIG. 10(B) is a schematic representation of binding of WP to different sizes of T-AuNP;

FIG. 10(C) is the change in fluorescence of DNA walker for TPs of different sizes with reaction time (same orbital chain concentration);

FIG. 10(D) is the reaction rate of DNA walker in the presence of TPs of different sizes (same orbital chain concentration).

FIG. 11 is a graph showing the result of the response of DNA walker to ZIKV-RNA in example 1;

wherein, FIG. 11(A) is a fluorescence emission spectrum of the DNA walker moving relatively among particles in response to different concentrations of ZIKV-RNA; 0nM, 1nM, 2nM, 4nM, 5nM, 6nM, 8nM, 10nM, 12nM and 15nM, respectively, from a to j; the interpolated plot shows the linear relationship between the fluorescence signal and the concentration of ZIKV-RNA;

FIG. 11(B) is the specificity of the DNA walker in relative movement between the particles.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, aiming at the defects in the prior art, the invention provides a DNA walker with relative movement among particles, a preparation method of the walker and application of the walker in RNA nucleic acid fragment detection.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.

Example 1

1.1 Experimental materials and reagents

All DNA, RNA, Tris base, boric acid, ethylenediaminetetraacetic acid (EDTA), 40% (w/v) acrylamide/bisacrylamide solution (19: 1), Dithiothreitol (DTT) and diethyl pyrocarbonate water (DEPC water) were purchased from shanghai bio-technology limited (china, shanghai).

Chloroauric acid trihydrate (HAuCl)₄·3H₂O), sodium citrate dihydrate (Na)₃C₆H₅O₇·2H₂O) and tris (2-carboxyethyl) phosphine (TCEP) were purchased from Sigma-Aldrich (St. Louis, USA).

5nm and 25nm gold nanoparticles (AuNPs) were purchased from Nanjing Xiancheng nanomaterial technology Co., Ltd, China, Nanjing. The chemicals used in the experiment were all analytically pure and no further purification was required during use. All solutions were prepared using DEPC water. The DNA and RNA sequences in this work are listed in table 1.

Table 1. DNA and RNA sequences used in this work.

Note: the underlining of the walking chain represents the catalytic sequence of 8-17E DNAzyme. The underlined rA of the orbital strand indicates adenine ribonucleotide.

1.2 Experimental instruments

The ultraviolet-visible absorption spectrum was obtained by using a UV-2910 ultraviolet spectrophotometer (Hitachi, Japan). Transmission Electron Microscope (TEM) images were obtained on a JSM-6700F transmission electron microscope (JEOL, Japan) at a voltage of 200 kV. The fluorescence spectrum was measured on an F-7000 fluorescence spectrophotometer (Hitachi, Japan). The excitation wavelength was set at 488nm and the emission wavelength ranged from 510-650 nm. The slit width for excitation and emission was set to 10nm and the voltage of the photomultiplier tube was 700V. Polyacrylamide gels by GelDocTM XR⁺Imaging systems (berle corporation, usa) image. Dynamic Light Scattering (DLS) measurements were performed on a nanometer particle sizer (british, marvens).

2. Preparation and characterization of DNA walker with relative movement among particles

And synthesizing 13nm-AuNPs by referring to the existing method. Sodium citrate solution (5mL, 38.8mM) was added rapidly to boiling HAuCl which was vigorously stirred₄After boiling continuously for 10 minutes in an aqueous solution (50mL, 1mM), the mixture was stirred continuouslyFor 15 minutes. The solution was cooled to room temperature with continuous slow stirring, filtered through a 0.22 μm filter and stored in a refrigerator at 4 ℃ protected from light. The AuNPs are characterized by an ultraviolet spectrophotometer and a transmission electron microscope, and the concentration of the synthesized 13nm AuNPs is determined according to the absorbance at 519nm and the corresponding molar absorption coefficient (2.7 multiplied by 10)⁸L mol^-1cm^-1) And (4) determining.

DNA walker consists of WPs and TPs. To prepare WPs, the walking and closed chains were linked in a 1: 5 in 1 XTris-HCl buffer (pH 8.3). The mixture was heated to 90 ℃ for 10 minutes and then slowly cooled to room temperature to form a closed walking chain. Prior to functionalization, thiolated DNA was mixed with TCEP at a ratio of 1: 100 for 2 hours to reduce the disulfide bonds of the DNA. Next, a mixture containing 50. mu.L of blocked walking chain (30. mu.M) and 1mL of AuNP (10nM) was stirred slowly at room temperature for 16 hours. Then, 100. mu.L of 2M NaCl was gradually added to the above mixture in six divided portions at 40-minute intervals. After continuing to stir slowly for 24 hours, the solution was centrifuged at 12,500rpm for 30 minutes to separate WPs from the unfunctionalized DNA. WPs were washed 3 times in Tris-HCl buffer (pH 8.0) and dispersed in 1 × Tris-HCl buffer (10mM Tris, pH 8.3).

To prepare TPs, a mixture containing 20. mu.L of TCEP reduced orbital chain (100. mu.M) and 1mL of AuNP (10nM) was stirred slowly at room temperature for 16 h. TP is then obtained by the same procedure as described above, including the steps of adding salt, separating and washing. Finally, TPs were dispersed in Tris-HCl buffer (10mM Tris, pH 8.3). Prepared WPs and TPs were stored in a refrigerator at 4 ℃ in the dark, WPs and TPs were characterized by UV-visible spectrophotometer and concentrations were determined.

3.1 determination of the amount of DNA strands on 3.1WPs and TPs

To measure the number of DNA strands per AuNP of WPs and TPs, DTT was first used to release DNA strands from aunps into solution, in accordance with the literature reported DTT substitution method. WPs and TPs were mixed separately with 20mM DTT and incubated overnight at room temperature. The solution was then centrifuged at 12,000rpm for 15 minutes to precipitate AuNPs, and the supernatant containing the released DNA strands was tested for fluorescence. The concentration of DNA strands was measured by using the fluorescence of the supernatant and a calibration curve of fluorophore-labeled DNA strands. Finally, the number of modified DNA strands on each AuNP of WPs and TPs was determined by dividing the concentration of the DNA strands by the concentration of the AuNP.

3.2 Experimental procedures for running DNA walker in relative motion between particles

For a typical inter-particle relative motion DNA Walker run, first, 6. mu.L of the target sequence (250nM) was added to a DNA Walker containing 12. mu.L of WPs (1nM), 12. mu.L of 5 × Tris-HCl (100mM Tris-HCl, 875mM NaCl, pH 8.3), 12. mu.L of MgCl₂(50mM) and 6. mu.L DEPC-H₂O in solution. The above mixed solution was incubated at 37 ℃ for 1.5 hours to expose free walking strands at WPs, and then 12. mu.L of TP (5nM) was added to initiate the walking of DNA Walker. The mixed solution was reacted at 37 ℃ for 2.5 hours. After completion of the DNA walker run, the above solution was centrifuged at 12,000rpm for 15 minutes, and then the fluorescence of the supernatant was measured.

3.3 gel electrophoresis analysis

The feasibility of the DNA walker run with relative movement between particles was demonstrated by using 15% native polyacrylamide gel electrophoresis (PAGE) experiments. First, the mixture contained 1. mu.L of blocked walking chain (10. mu.M), 1. mu.L of target sequence (10. mu.M), 1. mu.L of orbital chain (10. mu.M), 2. mu.L of 5 × Tris-HCl (pH 8.3), and 2. mu.L of MgCl₂(50mM) and 3. mu.L DEPC-H₂O10 u L reaction solution at 37 degrees C were incubated for 1 h. Subsequently, 2. mu.L of 6 × loading buffer was added to the above solution, and the mixture was loaded on 15% polyacrylamide gel to separate DNAs of different molecular weights. Next, the gel was run at 30mA constant current for 65 minutes at 15 ℃ using 1 XTBE buffer (89mM Tris, 89mM boric acid, 2mM EDTA, pH 8.3). The gel was then stained with SYBR Gold for 40 minutes and imaged by a gel imaging system.

4.1 characterization of synthetic 13nm gold nanoparticles

Ultraviolet visible absorption spectroscopy and Transmission Electron Microscopy (TEM) imaging were used to characterize the synthetic 13 nm-AuNPs. As can be seen from FIG. 2A, the UV-vis absorption spectrum shows that the maximum absorption wavelength of AuNPs is 519 nm. And fig. 2B shows TEM images showing that the shape of the synthesized AuNPs is circular, the particles are uniformly distributed, and the size is about 13 nm. These results indicate that 13nm AuNPs were successfully prepared.

4.2 characterization of 4.2WPs and TPs

As shown in FIG. 3A, the UV-vis characterization shows that the maximum absorption peak of the gold nanoparticles is 519nm, and the maximum absorption peaks of WPs and TPs are red-shifted to 522nm after the DNA chain is modified, which indicates that the DNA chain is functionalized on AuNPs. As shown in FIG. 3B, a standard curve was constructed based on the measured walking chain fluorescence of different concentrations of FAM-labeled FAM, and the resulting standard equation was F86.6 +73.5C_DNA. And substituting the fluorescence intensity of WPs measured after DTT substitution into a standard equation to obtain the concentration of the walking chains, and calculating the number of the modified walking chains on each WP to be 21. Similarly, the number of modified track chains per TP was 88 per FIG. 3C.

5.1 feasibility study

To verify the feasibility of the walk run of DNA with relative movement between particles, fluorescence characterization was first performed. As shown in FIG. 4A, in the presence of the target sequence, a significantly enhanced fluorescence signal was obtained. This indicates that the target sequence can hybridize to the closed strand and expose the DNAzyme sequence on the walking strand and further trigger the running of DNA walker. In contrast, in the absence of the target sequence, only a weak fluorescent signal was obtained, indicating that the DNAzyme sequences on the walking strand were blocked and unable to trigger DNA walker operations. When only TPs were present, a weaker fluorescence signal was obtained, indicating that the tracks on the TPs were stable unless WPs for walking and cleavage was present. When Mg is not present²⁺When the fluorescence intensity was almost the same as that of the background, it was shown that WPs was due to the lack of Mg²⁺Cannot walk along the TP as a cofactor for the DNAzyme cleavage reaction. Next, PAGE experiments were performed for verification. As shown in FIG. 4B, it can be concluded from lane 7 that a strand displacement reaction occurs between the target and the blocked walking strand, as compared to the single DNA markers of lanes 1 to 6. Wherein the target sequence hybridizes to the blocked strand to form a duplex with release of the walking strand. When the target sequence was not present, a band corresponding to the orbital chain was observed (lane 8). This indicates that the DNAzyme sequence on the walking chain is blocked and does not trigger the DNAzyme cleavage reaction. When the target sequence is presentAt this time, the band corresponding to the orbital strand disappears (lane 9), indicating its cleavage by the walking strand and the specific initiation of walker by the target. All results demonstrate the feasibility of the designed DNA walker run.

5.2 kinetic study of the relative movement of the particles of DNA walker in response to different concentrations of the target

To better assess WPs the effect of the polypod walking chain on walker performance, the kinetics of DNA walker in response to different target concentrations was studied. As shown in FIG. 5A, the fluorescence intensity response gradually increased from 0nM to 25nM target concentration with increasing reaction time. The change in the fluorescence signal of DNA walker rapidly increased in the first 30 minutes of the reaction, followed by a slow increase (0.5-2.5 hours) and finally stabilized (2.5-3.5 hours). This is because the walking chain can initially interact with a large number of orbital chains on the TP, and therefore exhibits a significant fluorescence increase within the first 30 minutes. As the DNA walker runs from 0.5 hour to 2.5 hours, the number of the walking regions and the orbital chains on TPs gradually decreases, and the change of fluorescence shows a slowly increasing trend. The change in fluorescence gradually stabilized after 2.5 hours of reaction as the orbital chain concentration tended to deplete. By measuring the fluorescence intensity at 5 minute intervals during the first 30 minutes of the reaction, a good linear relationship was obtained (fig. 5B). The fluorescence intensity of walker increases linearly with increasing target concentration. Then, the initial rate of DNA walker was determined by calculating the concentration of the orbital chain consumed per unit time in the first 30 minutes (FIG. 5C). Within the first 30 minutes of the reaction, the initial rate V of DNA wallker increased from 2.5X 10 with the target concentration increased from 5nM to 25nM^-12Increased to 1.2 × 10^-11M s^-1. This is because the gradual number of released travel chains at WPs increases with increasing target concentration. As the orbital chain is consumed, the increased free-running chain on the WP can more easily bind the residual orbital on the TPs, so that the forward running of the WP and the relative motion of the WP along the TP are induced, the running speed of the DNA walker is increased, and more fluorescent signals are generated.

5.3 comparison of the Performance of the DNA walker moving relatively between particles with that of the DNA walker moving in particles

To confirm more intuitivelyThe walking speed and the continuous running capability compare the fluorescence signals of the traditional DNA walker running in the particles and the DNA walker relatively moving between the particles. As a result, as shown in FIG. 6A, the DNA walker moving relatively between particles showed a rapid and enhanced increase in fluorescence signal. For comparison, control intra-particle-running DNA walkers were obtained by co-modifying the closed walking and orbital strands to the same AuNPs, wherein the modification ratio between the DNA strands was the same as the inter-particle-relative-movement DNA walkers, while the concentration of the orbital strands used was consistent with that used when modifying TPs. The fluorescence change of the DNA walker running in the particles was tested under the same experimental conditions as the DNA walker moving relatively between the particles, showing a slowly increasing fluorescence signal. The reaction rate of the first 30 minutes, the DNA walker of the relative movement among particles (1.21X 10) are obtained by calculation^-11M s^-1) The reaction rate of (2) is higher than that of DNA walker (5.91X 10) running in the pellet^-12M s^-1) The reaction rate of (2) is increased, which shows that the DNA walker moving relatively among particles has an accelerated walking speed (FIG. 6C). When the reaction time is about 1h, the DNA walker running in the particles has no obvious fluorescence increase, and the DNA walker relatively moving among the particles still shows a continuously increased fluorescence signal within 2.5h, as shown in FIG. 6B, the signal accumulation degree of the two types of DNA walkers can be intuitively obtained from the fluorescence spectrum when the reaction reaches the platform. The DNA walker moving relative to each other among the particles obtained a 2.1-fold increase in the accumulated signal compared to the DNA walker moving inside the particles. In addition, according to the corresponding fluorescence intensity and the standard curve of the FAM-labeled track chain, the concentration of the track chain released in the running process of the DNA walker is calculated, and then the ratio of the concentration of the track chain released on the TPs to the concentration of the total track chain is calculated. The result shows that 52% of the orbital chains are released during the walking process of the DNA walker moving relatively among the particles, and 31% of the orbital chains are released during the walking process of the DNA walker moving in the particles. In the process of walking the DNA walker in the relative movement among the particles, after the WP finishes walking along the surface of one TP, the WP can continuously search for a new TP to carry out the relative movement, so that the walking area is enlarged, and a rapidly enhanced fluorescence signal is obtained.

5.4 Effect of TPs of different sizes on the running performance of the DNAwalker in relative movement between particles

This example investigates the effect of AuNPs size variation in TPs on DNA walker performance. First, orbital chains were modified on AuNPs of different sizes (5nm, 13nm and 25nm), wherein the 5nm and 25nm AuNPs were also characterized by UV-visible spectroscopy and TEM (FIG. 7), to obtain TP-5nm AuNPs (TP-Au5), TP-13nm AuNPs (TP-Au13) and TP-25nm AuNPs (TP-Au 25). Wherein the modification amounts of the orbital chains on TP-Au5 and TP-Au25 were measured at 17 and 185, respectively (FIG. 8). Dynamic Light Scattering (DLS) characterization gave hydrated diameters of TP-Au5, TP-Au13, TP-Au25 and WP of 26.0nm, 37.8nm, 32.2nm and 43.8nm, respectively (FIG. 9). Among them, although the diameter of AuNPs in TP-Au25 is larger, the orbital chain is in a "lying" conformation on TP-Au25, which is probably due to the difference in the density of orbital chains on AuNPs, and the density of orbital chains on TP-Au25 (9.4X 10)¹²/cm²) Much less than the density of the orbital chains on TP-Au13 (1.7X 10)¹³/cm²) This indicates a "lying" conformation of TP-Au25 and a smaller hydrated diameter. As shown in fig. 10A, the fluorescence of DNAwalker was changed depending on the size change of TP at the same concentration of TP. By treating WP and TP as two colliding particles, these problems can be explained by the difference in collision efficiency between WPs and the size of the TP around them. The collision efficiency of particles of different particle sizes is smaller than that of particles of the same particle size, and the larger the ratio of the radii of the two colliding particles is, the smaller the collision efficiency is. Ratio of radii of two particles in the reaction solution of TP-Au5, TP-Au13 and TP-Au25 (R)_WP/R_TP) 1.68, 1.15 and 1.36 respectively. A schematic diagram of combinations of WP and different sized TPs is shown in fig. 10B. When TP-Au13 was present in the reaction solution, WPs and TPs in the DNA walker system had similar particle sizes and produced the strongest change in fluorescence signal. When TP-Au5 and TP-Au20 are present in the system, the difference in particle size between WPs and TPs is large, and therefore collision efficiency is reduced. The ratio of the radius of WPs to the radius of TPs in the TP-Au5 system is larger than that of the TP-Au25 system, and the modification amount of the orbital chain on the TP-Au5 is the least, so that the TP-Au5 shows the weakest fluorescence signal.

This example further investigated the distribution of the same number of orbital strands over which size of TPs is beneficial for achieving efficient operation of DNA walker. In order to maintain the same orbital chain concentration, the molar ratios of WP to TP-Au5, TP-Au13 and TP-Au25 in the reaction solution were adjusted to 1: 25.8, 1: 5 and 1: 2.3. as shown in FIG. 10C, TP-Au13 showed the fastest signal growth and obtained the highest fluorescence intensity because WP and TP-Au13 in the reaction solution had similar particle sizes. Although the collision efficiency between WPs and TPs in the TP-Au5 reaction solution is generally weaker than that of TP-Au25, the concentration of TP-Au5 is higher, and thus WP may be combined with surrounding pluralities of TP-Au5 to generate accelerated reaction rate and enhanced fluorescence signal, compared to TP-Au25 (fig. 10D). The results show that arranging the orbital chains on the surface of a particle of approximately WPs size is advantageous for increasing the walking speed and signal accumulation of such inter-particle DNA walker.

5.5 detection Performance of inter-particle relative movement DNA walker

Zika virus (ZIKV) is a single-stranded positive strand RNA virus of the flaviviridae family. ZIKV infection causes high morbidity and mortality rates pose a significant threat to human health worldwide, and no effective drugs or licensed vaccines are available to date. The key to the detection of pathogenic viruses is high sensitivity, and early discovery and detection contribute to improving survival rate. Different concentrations of ZIKV-RNA were assayed under optimal experimental conditions to evaluate the detection performance of DNA walker. As shown in FIG. 11A, the fluorescence intensity gradually increased with the increase in the concentration of ZIKV-RNA. The calibration curve in the inset shows that fluorescence intensity shows a good linear relationship with ZIKV-RNA concentration in the range of 1nM to 15 nM. The correlation coefficient obtained from the linear equation shows good correlation over the concentration range of ZIKV-RNA. The estimated limit of detection (LOD) was 118pM according to the triple standard deviation principle (LOD 3 σ/K, where σ is the standard deviation of blank replicates and K is the slope of the calibration curve). Furthermore, the proposed sensitivity of DNA walker to ZIKV-related nucleic acid sequences was higher than some reported analytical strategies (table 2).

To examine the specificity of this DNA walker, this example compared the RNA sequences of the target ZIKV (T-ZIKV), dengue virus (T-DENV), Japanese encephalitis virus (T-JEV) and yellow fever virus (T-YFV) under the optimal experimental conditions. These four viruses all belong to the flaviviridae family, the genus flavivirus single-stranded RNA virus, and have similar clinical symptoms. Under the same conditions, only the target RNA sequence caused a significant fluorescence enhancement, while the RNA sequences of other similar viruses only caused a fluorescence signal close to the blank (FIG. 11B). The result shows that the DNA walker has good specificity for the detection of the ZIKV-RNA.

TABLE 2 comparison of DNA walker with some reported ZIKV related sequence detection strategies

5.6 analysis in Complex biological samples

In order to investigate the practical application capability of the inter-particle relative motion DNA walker in a complex biological sample, a standard recovery experiment is carried out by taking human serum as a complex system. Adding ZIKV-RNA with different concentrations into human serum, detecting the sample by using DNA walker, and then determining the standard recovery rate of the ZIKV-RNA in the human serum. According to the table 3, the recovery rate of the ZIKV-RNA in the human serum is between 97.5% and 103.5%, which shows that the DNA walker can effectively resist the interference of the coexisting complex components in the human serum. The good anti-interference ability is attributed to the stability of WPs and TPs in the reaction solution, because the AuNPs on WPs and TPs have strong space effect and high ionic charge, and can be used for effectively stabilizing DNA. In addition, the results also prove that the DNAwalker has potential application in the analysis of actual biological samples.

TABLE 3 labeling recovery experiment of ZIKV-RNA in human serum

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong university

<120> DNA walker for relative movement between particles and application of biosensing thereof

<130> 201927610

<160> 3

<170> PatentIn version 3.3

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<211> 69

<212> DNA

<213> Artificial sequence

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tttttttttt tttttttttt tttttttttg ggaaagactc tcttctccga gccggtcgaa 60

atagtgcgt 69

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<212> DNA

<213> Artificial sequence

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aagagagtct ttcccacgtc aatatgct 28

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<212> DNA

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tttttttttt ttttacgcac tatraggaaga gat 33

Claims

1. The DNA walker with the relative movement among the particles is characterized by comprising walking particles and track particles, wherein the walking particles are composed of first gold nanoparticles and closed walking chains, the walking chains are single-stranded DNA and comprise DNAzyme sequences, one ends of the DNAzyme sequences are connected with the gold nanoparticles, and the other ends of the DNAzyme sequences are combined with the closed chains; the closed strand comprises a complementary sequence of a target to be detected;

2. The DNA walker of relative inter-particle motion of claim 1 wherein the walking chain comprises the sequence of 8-17E DNAzyme.

3. The DNA walker with relative motion between particles as claimed in claim 1, wherein the first gold nanoparticle or the second gold nanoparticle has a particle size of 10-15 nm.

4. The DNA walker with relative motion between particles as claimed in claim 1, wherein the first gold nanoparticles or the second gold nanoparticles are HAuCl coated₄Is prepared by a citric acid reduction method.

5. The method for preparing the DNA walker with the relative motion among the particles as claimed in any one of claims 1-4, wherein the preparation method comprises the following steps:

6. The method for preparing the DNA walker with the relative motion among the particles as claimed in claim 5, wherein the ratio of the walking chain to the closed chain is 0.8-1.2: 4-6.

7. The method for preparing the DNA walker with the relative motion among the particles as claimed in claim 5, wherein the walking chain and the closed chain are mixed and heated to 88-92 ℃.

8. The method for preparing the DNA walker with the relative motion among the particles as claimed in claim 5, wherein the molar ratio of the closed walking chain to the first gold nanoparticles is 140-160: 1; the molar ratio of the rail chain to the second gold nanoparticles is 190-210: 1.

9. Use of the DNA walker in the relative movement between particles as claimed in any one of claims 1 to 4 in the preparation of a nucleic acid biosensor.

10. A nucleic acid biosensor, wherein the sensor comprises the DNA walker for the relative movement between particles according to any one of claims 1 to 4.

11. The nucleic acid biosensor according to claim 10, wherein the detection method of the nucleic acid biosensor is as follows: the sequence to be detected, the walking particles and MgCl₂Adding the mixed solution into a buffer solution to obtain a mixed solution, incubating for a period of time, adding the rail particles, reacting, and centrifuging to detect the fluorescence in the supernatant after reacting for a period of time.

12. The nucleic acid biosensor in accordance with claim 11, wherein the buffer solution is a Tris-HCl buffer solution containing NaCl at a pH of 8 to 9.

13. The nucleic acid biosensor in accordance with claim 11, wherein the mixed solution is incubated at 35-39 ℃ for 1-2 hours.

14. The nucleic acid biosensor in accordance with claim 11, wherein the reaction time is 2 to 3 hours.

15. A nucleic acid detection kit comprising the DNA walker in relative movement between particles according to any one of claims 1 to 4.