阅读说明：本技术 一种纳米笼探针及其应用和核酸检测方法 (Nano cage probe, application thereof and nucleic acid detection method ) 是由 李美星 程娟 沈清明 范曲立 于 2020-05-25 设计创作，主要内容包括：本发明公开了一种纳米笼探针及其应用和核酸检测方法,包括金/银纳米笼颗粒,颗粒的表面修饰有发卡DNA链,发卡DNA链与待测核酸部分互补；并以金/银纳米笼颗粒作为核酸识别反应基底和散射信号指示探针,在探针表面修饰发夹链作为核酸识别单元,利用DNA碱基互补配对和链取代反应规则,实现DNA循环和HCR扩增过程,从而在探针表面形成G-四链体-血红素DNA酶,通过酶催化过氧化氢分解产生的活性氧实现对纳米笼中银组分的刻蚀,引起暗场信号的“灭灯”响应。这种具有时间分辨能力的暗场信号改变,显示出对待测核酸浓度的依赖性,并结合统计分析方法,进行定量分析。(The invention discloses a nano cage probe and application thereof and a nucleic acid detection method, comprising gold/silver nano cage particles, wherein the surface of the particles is modified with hairpin DNA chains, and the hairpin DNA chains are partially complementary with nucleic acid to be detected; gold/silver nanocage particles are used as a nucleic acid recognition reaction substrate and a scattered signal indicating probe, a hairpin chain is modified on the surface of the probe and used as a nucleic acid recognition unit, DNA base complementary pairing and chain substitution reaction rules are utilized to realize DNA circulation and HCR amplification processes, so that G-quadruplex-heme DNase is formed on the surface of the probe, active oxygen generated by enzymatic hydrogen peroxide decomposition is used for etching silver components in the nanocage, and lamp-off response of a dark field signal is caused. The dark field signal with time resolution changes, shows the dependence on the concentration of the nucleic acid to be detected, and is combined with a statistical analysis method for quantitative analysis.)

1. A nanocage probe, characterized in that: the kit comprises gold/silver nanocage particles, wherein hairpin DNA chains are modified on the surfaces of the particles and are partially complementary with nucleic acid to be detected; the particles are of a hollow structure, the outer wall of each particle is made of gold, and the inner wall of each particle is made of silver.

2. The nanocage probe of claim 1, wherein: the gold/silver nanocage particles are prepared by a method of reducing a silver ball template by chloroauric acid, and the particle size of the particles is 40-100 nm.

3. Use of the nanocage probe of claim 1 in the preparation of a nucleic acid detection kit or dark field imaging monitoring.

4. A method for detecting nucleic acids for non-disease diagnostic purposes, comprising the steps of:

(1) gold and silver nanocage particles are used as a nucleic acid identification reaction substrate and a scattering signal indication probe, and a hairpin DNA chain is modified on the surface of the probe to be used as a nucleic acid identification unit;

(2) realizing DNA circulation and HCR amplification process by using DNA base complementary pairing and strand displacement reaction rules, and forming G-quadruplex-heme DNase on the surface of the probe;

(3) etching of silver components in the nano cage particles is realized through active oxygen generated by enzymatic hydrogen peroxide decomposition, so that a dark field signal in dark field imaging is changed, and quantitative detection is performed by utilizing the relation between the change time of the dark field signal and the concentration of nucleic acid to be detected.

5. The detection method according to claim 4, characterized in that: punching a hole on the organic silicon film, and sticking the hole with positive charge glass to be used as a reaction tank and a detection tank; diluting the gold/silver nano cage particles prepared in the step (1), adding the diluted gold/silver nano cage particles into a pool, and dispersing and fixing the diluted gold/silver nano cage particles on the surface of positive charge glass to form a detection substrate.

6. The detection method according to claim 4, wherein the step (2) comprises: mixing the prepared probe with a sample solution containing a DNA probe H2 and nucleic acid to be detected for reaction, and washing a substrate by using a buffer solution; then adding a mixed solution of hairpin probes H3 and H4 for incubation, carrying out hybrid chain reaction and washing by using a buffer solution; adding hemin solution containing potassium ions, reacting, and cleaning with buffer solution containing potassium ions to obtain a nano cage probe with a surface forming G-quadruplex-heme DNA enzyme; wherein H2 is partially complementary to the hairpin DNA strand in step (1), H3 is partially complementary to the hairpin DNA strand and H2, and H4 is partially complementary to H3.

7. The detection method according to claim 4, characterized in that: the change time of the dark field signal is the waiting time from the addition of the detection liquid containing hydrogen peroxide to the particle lamp-out, and the particle lamp-out is the reduction of the gray value of the particles before and after the dark field intensity by 50-80%.

8. The detection method according to claim 5, characterized in that: after the gold/silver nanocage particles are immobilized, the detection substrate is incubated with 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution to neutralize excess positive charges on the glass surface.

9. The detection method according to claim 4, wherein the nucleic acid to be detected is microRNA or DNA, and the concentration of the nucleic acid to be detected is 1 × 10^-16～1×10^-13mol/L。

10. The detection method according to claim 6, characterized in that: the concentration of H2 is 10nM to 30nM, and the concentrations of H3 and H4 are 50nM to 150 nM.

Technical Field

The invention relates to a probe, application thereof and a nucleic acid detection method, in particular to a nano cage probe, application thereof and a nucleic acid detection method.

Background

The nucleic acid is one of the basic materials of life, carries important genetic information and strictly controls protein synthesis and gene expression, and the strong specificity of nucleic acid detection is ensured by the base complementary pairing principle followed by the nucleic acid in the process of identification and hybridization, so that the nucleic acid detection has important significance. However, when the content of nucleic acid is low, the sensitivity and accuracy of nucleic acid detection are difficult to meet with the large sample requirement and complicated pretreatment operation in conventional detection methods. Therefore, it is necessary to develop a sensitive and accurate nucleic acid detection method, thereby further shortening the detection window period.

Disclosure of Invention

The purpose of the invention is as follows: one of the objects of the present invention is to provide a nanocage probe having a nucleic acid recognition function; the other purpose of the invention is to provide the application of the nano cage probe; it is a further object of the present invention to provide a method for detecting nucleic acid, which can realize ultrasensitive detection of nucleic acid at low concentration.

The technical scheme is as follows: the nano cage probe comprises gold/silver nano cage particles, wherein hairpin DNA chains are modified on the surfaces of the particles and are partially complementary with nucleic acid to be detected; the particles are of a hollow structure, the outer wall of each particle is made of gold, and the inner wall of each particle is made of silver.

Wherein, the type of the nucleic acid to be detected is microRNA or DNA. The preparation method of the nano cage probe comprises the steps of preparing a silver ball template, synthesizing a gold/silver nano cage and functionalizing nucleic acid on the surface of the nano cage.

Preferably, the gold/silver nanocage particles are prepared by a method of reducing a silver ball template by chloroauric acid, and the particle size of the particles is 40-100 nm. Optionally, in the embodiment, a particle preparation process with a particle size of 52 ± 5nm is shown, and the synthesized particle size may be within a range of 40-100 nm.

Preferably, G-quadruplex-2-erythrose DNase is formed on the surface of the nano-cage probe and is used as a signal sensor and a signal amplifier for detection.

The invention provides application of the nano cage probe in preparation of a nucleic acid detection kit or dark field imaging monitoring. The gold nano cage particles are used as a reaction substrate and a scattering signal indicator, a nucleic acid identification and sensing probe is constructed on the surface of the gold nano cage particles, and the time resolution of the scattering signal is realized by utilizing the sensitive response of the nano cage particles to active oxygen in the environment, so that the gold nano cage particles are used for the ultra-sensitive detection of nucleic acid.

The invention realizes the real-time monitoring of the dynamic reaction process by the dark field imaging technology of the metal nano particle Localized Surface Plasmon Resonance (LSPR). The method takes gold/silver nano cage particles modified with hairpin DNA as a plasma probe, constructs a nucleic acid identification unit on the surface of the plasma probe, and realizes the DNA circulation and hybridization amplification process by utilizing the base complementary pairing and chain substitution reaction rules of the DNA. In heme (hemin) and K⁺And G-quadruplex-heme DNase is formed in the presence of ions, and the etching of the silver component in the nanocage is realized by catalyzing active oxygen (ROS) generated by decomposing hydrogen peroxide through enzyme catalysis. The etching process causes the content of silver in the silver/gold nanometer cage structure to gradually decrease along with the lapse of etching time, so that a dark field scattering signal of the single-particle nanometer probe suddenly disappears, and a 'light-out' response appears. The etching rate of the micro-RNA is directly related to the content of the target microRNA, and the micro-RNA is represented as dark field signal change based on a time dimension. The concentration determination of the microRNA-21 can be realized by combining a statistical analysis method. The constructed sensor has higher sensitivity and selectivity, and the sensitive real-time response characteristic of the sensor confirms that the nano cage particles can be used as probes for surface/interface reactions and indicators for microenvironment change, thereby showing the application prospect of the nano cage particles in dark-field biosensing and life analysis.

The invention also provides a nucleic acid detection method for non-disease diagnosis purposes, comprising the following steps:

(1) taking gold/silver nano cage particles as a nucleic acid recognition reaction substrate and a scattering signal indication probe, and modifying a hairpin DNA chain on the surface of the probe to be used as a nucleic acid recognition unit;

(2) realizing DNA circulation and HCR amplification process by using DNA base complementary pairing and strand displacement reaction rules, and forming G-quadruplex-heme DNase on the surface of the probe;

(3) etching of silver components in the nano cage particles is realized through active oxygen generated by enzymatic hydrogen peroxide decomposition, so that a dark field signal in dark field imaging is changed, and quantitative detection is performed by utilizing the relation between the change time of the dark field signal and the concentration of nucleic acid to be detected.

And (3) taking target nucleic acid to be detected with different concentrations, detecting the target nucleic acid to be detected after the same operation is carried out, and obtaining a relation curve graph between the light-out time (change time of dark field signals) of the nano cage probe and the concentration of the nucleic acid to be detected.

Preferably, a hole is punched in the organic silicon film, and the organic silicon film is pasted with positive charge glass to be used as a reaction tank and a detection tank; diluting the gold/silver nano cage particles prepared in the step (1), adding the diluted gold/silver nano cage particles into a pool, and dispersing and fixing the diluted gold/silver nano cage particles on the surface of positive charge glass to form a detection substrate. Optionally, the organic silicon film is a polydimethylsiloxane polymer film; the positively charged glass means that the glass surface has a positive charge.

Preferably, step (2) comprises: mixing the prepared probe with a sample solution containing a DNA probe H2 and nucleic acid to be detected for reaction, and washing a substrate by using a buffer solution; then adding a mixed solution of hairpin probes H3 and H4 for incubation, carrying out hybrid chain reaction and washing by using a buffer solution; adding hemin solution containing potassium ions, reacting, and cleaning with buffer solution containing potassium ions to obtain a nano cage probe with a surface forming G-quadruplex-heme DNA enzyme; wherein H2 is partially complementary to the hairpin DNA strand in step (1), H3 is partially complementary to the hairpin DNA strand and H2, and H4 is partially complementary to H3.

Preferably, the concentration of H2 is 10nM to 30nM, and the concentrations of H3 and H4 are 50nM to 150 nM.

Preferably, after the gold/silver nanocage particles are immobilized, the detection substrate is incubated with 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution to neutralize excess positive charge on the glass surface.

Preferably, the dark field signal is varied in time from the addition of H-containing₂O₂The waiting time from the detection liquid to the particle lamp-out is that the gray value of the particle before and after the dark field intensity is reduced by 50-80%; a visual detection process can be implemented.

Preferably, the nucleic acid to be detected is microRNA or DNA, and the concentration of the nucleic acid to be detected is 1 × 10^-16～1×10^-13mol/L; too high a concentration can cause signals to appear too fast, and the waiting time is too short to distinguish; too low a concentration can result in too long a dark field detection time.

Further, the nucleic acid detection method comprises the following steps:

s1: preparing a gold/silver nanocage structure by a method of reducing a silver ball template by chloroauric acid; mixing and incubating the probe with hairpin DNA probe (H1) according to the concentration ratio of 1: 200 for 2 hours, and then aging, centrifuging and washing to obtain the plasma probe.

S2: and punching a hole on the polydimethylsiloxane polymer film, and sticking the hole with positive charge glass to be used as a reaction pool and a detection pool. Diluting the prepared gold/silver nanocage particles by 1000 times, adding 20 mu L of the diluted gold/silver nanocage particles into a pool, and dispersing and fixing the particles on the surface of positive charge glass to obtain a detection substrate.

S3: mu.L of a sample solution containing 10nM of the DNA probe (H2) and 20fM of the target microRNA or DNA is added to the well, after 1 hour of reaction, the substrate is washed with 0.1M phosphate buffer (PBS, pH 7.4), 20. mu.L of a mixture solution containing 50nM hairpin probes (H3 and H4) is added thereto, incubation is carried out for 1 hour, hybridization reaction is carried out and washing is carried out with 0.1M PBS buffer, and 50. mu.M hemin solution (containing 0.1M K) is added thereto⁺) After 30 minutes, the cells were washed with PBS containing 0.1M potassium ion. At this time, G-quadruplex-heme DNase is formed on the surface of the nano-cage probe particles.

S4: 20 μ L of 20mM H was added to the reaction cell₂O₂The phosphate buffer solution is used as a dark field detection solution, and then the dark field detection solution is observed under a dark field microscope and is photographed and monitored, and the change condition of a dark field signal along with time is recorded.

S5: and analyzing the imaging condition of the particles in the field to obtain the dark field signal 'light-off' response time of the single particle. Through statistical analysis, a standard curve between the response time of 'light-off' and the concentration of the nucleic acid to be detected is obtained.

S6: monitoring the plasma probe at H after incubation with unknown concentrations of nucleic acid to be detected₂O₂Detecting dark field signals in the liquid, performing statistical analysis, and estimating by combining a standard curve to obtain the concentration of the nucleic acid to be detected in the liquid to be detected.

In the step S1, the gold/silver nanocages have a particle size of 40-100 nm, are hollow inside, and have silver as an inner wall component and gold as an outer wall component; the sequence of hairpin DNA modified on the surface is partially complementary with the sequence of the microRNA or DNA of the target object.

In step S2, the inner diameter of the circular hole of the reaction cell was 3mM, and after the plasma probe was immobilized, incubation was performed for 30 minutes using 2mM SMCC solution (4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution) to neutralize the excess positive charge on the glass surface.

In step S3, after each incubation, washing was performed using PBS buffer (containing 0.1M KCl) with pH 7.4.

In step S5, in dark field imaging, a "light-off" process in which particles are darkened can be observed with time, and according to the extracted gray value information in the image, the gray value before and after the dark field intensity of the particles is reduced by about 67% ± 13%.

In step S5, the response time is determined from the content of H₂O₂The waiting time of the detection liquid added to the particle 'light-off' is counted, the waiting time of at least 120 particle dark field responses is counted, and fitting is carried out by Gaussian distribution to obtain the statistical result of the average waiting time; and drawing a relation graph between the nucleic acid to be detected with different concentrations and different average waiting times to obtain a standard curve.

In step S6, a nucleic acid solution to be tested containing unknown concentration is incubated with a plasma probe, the same subsequent processing is performed, the waiting time of more than 100 particles is counted, and gaussian distribution is used for simulation to obtain the average waiting time. And (5) comparing with the standard curve, and estimating the concentration of the nucleic acid to be detected in the liquid to be detected.

The invention also provides the application of the analysis method in the detection of microRNA in a simple solution; simple solution refers to PBS buffer containing microRNA, containing no other interfering substances.

The invention principle is as follows: the dark field imaging method based on the metal nano particles has high response speed, no light bleaching and signal flicker, can realize time/space resolution and long-time-range observation in vivo, and has unique application prospect. Particularly gold and silver nanoparticles, exhibit a very high sensitivity to environmental media due to the Localized Surface Plasmon Resonance (LSPR) effect on the particle surface driven by the incident electromagnetic field. Therefore, the change (wavelength shift or intensity change) of the scattering signal of the nano-particles caused by biological recognition or chemical reaction on the surface and interface of the particles can be directly observed through a dark-field microscope, so that the real-time monitoring of the dynamic reaction process is realized. A nucleic acid detection system with high sensitivity and selective response is constructed by combining a DNA chain substitution or enzyme-assisted circulation strategy and a signal amplification technology, so that the signal resolution and response sensitivity can be further improved, the ultra-sensitive detection of a low-concentration nucleic acid target object is realized, and technical support is provided for nano-scale biosensing and extremely-low-content nucleic acid detection.

The invention takes gold/silver nanocage particles as a nucleic acid recognition reaction substrate and a scattered signal indication probe, modifies a hairpin chain on the surface of the probe as a nucleic acid recognition unit, realizes DNA circulation and HCR amplification processes by utilizing DNA base complementary pairing and chain substitution reaction rules, thereby forming G-quadruplex-heme DNA enzyme on the surface of the probe, realizes the etching of silver components in the nanocage by active oxygen generated by enzymatic hydrogen peroxide decomposition, and causes 'light-off' response of dark field signals. The dark field signal with time resolution changes, shows the dependence on the concentration of the nucleic acid to be detected, and is combined with a statistical analysis method for quantitative analysis.

The method takes gold nano cage particles as a reaction substrate and a scattering signal indicator, constructs a nucleic acid identification and sensing probe on the surface of the gold nano cage particles, utilizes the sensitive response of the nano cage particles to active oxygen in the environment, realizes the time resolution of the scattering signal, and is used for the ultra-sensitive detection of nucleic acid.

At K⁺In the presence of the ligand, a plurality of G-quadruplex-heme DNases are formed on the surface of the nano cage, hydrogen peroxide can be catalyzed to decompose to generate active oxygen (hydroxyl free radicals), silver components on the inner wall of nano cage particles are etched, so that the change of a single nano cage probe scattering signal is caused, and a 'light-out' response is displayed in dark field imaging.

The invention realizes the real-time monitoring of the dynamic reaction process based on the dark field imaging technology of the metal nano particle local surface plasma resonance; gold/silver nanocage particles are used as a plasma probe, and the processes of nucleic acid identification, DNA chain amplification, enzyme-like catalysis and the like constructed on the surface of the gold/silver nanocage particles finally trigger dark field lamp-off signals of the particles; the waiting time of the signal and the concentration of the nucleic acid to be detected present a better functional relationship, the resolution of time dimension can be clearly realized in the imaging process, and compared with the observation of the scattering spectrum displacement and the intensity change, the method is more intuitive and convenient for detection; the characteristics of real-time response and long-term observation of the device have outstanding advantages in continuous monitoring, and show the unique prospect of the single-particle dark-field imaging technology in biosensing and life analysis.

Has the advantages that: compared with the prior art, the method has the advantages that,

(1) the gold/silver nanocage particles are used as plasma probes, not only as substrates for nucleic acid identification and sensing, but also as indicator probes in the active oxygen etching process, and have sensitive response to active oxygen in the environment;

(2) the DNA hybridization and strand replacement process on the surface of the nano-cage probe has high specific recognition capability, and the recognition of the microRNA to be detected can initiate the subsequent DNA circulation and HCR amplification process, so that signal amplification is realized, and an effective amplification strategy is provided for the ultra-sensitive detection of the surface of a single particle;

(3) according to the invention, dark field 'light-out' response of the nano cage probe is taken as a monitoring signal, the waiting time of the light-out process and the concentration of an object to be detected present a better functional relationship, the resolution of time dimension can be clearly realized in the imaging process, compared with the observation of scattering spectrum displacement and intensity change, the method is more intuitive and convenient to detect, and the characteristics of real-time response and long-term observation show outstanding advantages in continuous monitoring;

(4) a time-resolved detection strategy depending on the concentration of a target object is constructed by combining a statistical method, and the time-resolved detection strategy is applied to the ultra-sensitive detection of microRNA-21, so that the unique prospect of a single-particle dark-field imaging technology in biosensing and life analysis is shown;

(5) the invention can realize the ultra-sensitive and visual rapid detection of the target nucleic acid at low concentration.

Drawings

FIG. 1 is a process for recognizing and triggering DNA chain substitution and formation of an enzyme-like structure of microRNA on the surface of a nano-cage probe;

FIG. 2 is an electron microscope representation and a high-resolution element distribution diagram of gold and silver nanocage particles; the picture at the upper left corner is an SEM picture, the picture at the upper right corner is a distribution diagram of Ag element, the picture at the lower left corner is a distribution diagram of Au element, and the picture at the lower right corner is a distribution superposition diagram of Ag and Au elements;

FIG. 3 shows the results of recognition of the nanocage probe with microRNA-21 of 20fM concentration in H₂O₂Dark field signal changes in the reaction solution;

FIG. 4 is a statistical distribution of the light-out time of 133 nanocage probes in a dark field and a Gaussian fitting curve thereof;

FIG. 5 is a graph showing the functional relationship between the time to extinguish the lamp and the concentration of microRNA-21 in the nanocage probe.

Detailed Description

The present invention will be described in further detail with reference to examples.

The materials and reagents used in the following examples are all commercially available; wherein, the microRNA-21 and microRNA-141 sequences are purchased from Shanghai Jima pharmaceutical technology, Inc., and other DNA sequences are purchased from biological engineering (Shanghai) company; polyvinylpyrrolidone (PVP) and chloroauric acid (HAuCl)₄·3H₂O), hydrogen peroxide (H)₂ O ₂30%), sodium hydroxide (NaOH), potassium chloride (KCl), disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from national drug group chemical Co., Ltd; AgNO₃Ascorbic Acid (AA) and hemin (hemin) were purchased from Aladdin reagent. Tris (2-carbonylethyl) phosphorus hydrochloride (TCEP), succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate sodium salt (sulfo-SMCC) was purchased from Sigma-Aldrich, Inc. The resistivity of the ultrapure water used in the experiment was 18.2 M.OMEGA.cm and was purified by a Milli-Q ultrapure water purifier. The PBS buffer was formulated to the desired concentration and pH from disodium hydrogen phosphate and sodium dihydrogen phosphate.