CN113736856A

CN113736856A - Isothermal nucleic acid amplification sensor for rapidly detecting hypochlorous acid and myeloperoxidase

Info

Publication number: CN113736856A
Application number: CN202111135010.2A
Authority: CN
Inventors: 孔德明; 汪晶; 马嘉懿; 王东霞; 刘博�; 井潇; 陈郸烨
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2021-09-27
Filing date: 2021-09-27
Publication date: 2021-12-03

Abstract

The invention provides an isothermal nucleic acid amplification sensor for rapidly detecting hypochlorous acid and myeloperoxidase, which comprises three parts: a, probe 1(P1) which is respectively composed of a plurality of DNA single strands (S1, S2 … Sn and H1) to form a three-dimensional rigid nano structure; b, probe 2(P2), which respectively consists of a plurality of DNA single strands (S1, S2 … Sn and H2) to form a three-dimensional rigid nanostructure, wherein H1 and H2 form two units of an amplification reaction such as a Hybrid Chain Reaction (HCR); c, probe 3(Lock-In), a phosphorothioate-modified nucleic acid constituting a chain (Lock), a primer chain (In) capable of initiating a hybridization chain reaction, wherein In is initially locked by Lock and fails to initiate HCR, and catalyzes a target Myeloperoxidase (MPO) when presentH₂O₂And chloride ion (Cl)^‑) Hypochlorous acid (HOCl) is generated, the phosphorothioate chain is cleaved, and In is subsequently released, which triggers the amplification reaction of H1 and H2. The nucleic acid nanoprobe can detect hypochlorous acid and myeloperoxidase.

Description

Isothermal nucleic acid amplification sensor for rapidly detecting hypochlorous acid and myeloperoxidase

Technical Field

The invention relates to the technical field of nano material synthesis and molecular detection, in particular to an isothermal nucleic acid amplification sensor for rapidly detecting hypochlorous acid and myeloperoxidase through DNA nano structure mediation.

Background

Pathological features of the disease are often accompanied by upregulation of certain biomarkers, such as ions, nucleic acids, small molecules, and proteins. The detection tool with high sensitivity and strong compatibility has important significance for accurate diagnosis of diseases. However, the sensitivity, accuracy and biosafety of probes remain a challenge, which has prompted researchers to explore more suitable materials and platforms to address these issues. Myeloperoxidase (MPO), a heme protease, is mainly derived from polymorphonuclear neutrophils and is also present in monocytes, macrophages. When the organism is activated by inflammation-stimulated phagocytosis, a large amount of active MPO is secreted into the phagocyte, catalyzing H₂O₂And chloride ion (Cl)^-) Generate hypochlorous acid (HOCl) with high bactericidal power. In addition to HOCl, MPO catalyzes the formation of other reactive molecules, such as aldehydes (aldehydes), tyrosine radicals (tyrosyl radials), hydroxyl radicals (· OH), and oxidizes Nitric Oxide (NO) to nitrite. Thus, MPO is associated with severe infections and various inflammatory diseases, such as cardiovascular disease, glomerular injury, rheumatoid arthritis, atherosclerosis, pulmonary fibrosis, alzheimer's disease, parkinson's disease and certain cancers. The determination of MPO plays a crucial role in the auxiliary diagnosis of the relevant diseases. Goldmann et al found that MPO was significantly elevated within 2 hours after onset of chest pain symptoms in patients with acute myocardial infarction, and that MPO was detected in the laboratory and widely used clinically in the judgment of myocardial cell damageThe cTNT of (A) needs to be increased 3-6 hours after the myocardial infarction occurs. Therefore, the research of MPOs and enzymatic reaction systems mediated thereby has received a great deal of attention, particularly in the fields of biology and medicine.

There are various methods for evaluating MPO activity, such as colorimetry, radiometry, electrochemistry, chemiluminescence, enzyme-linked immunosorbent assay, fluorescence imaging, magnetic resonance imaging, CT imaging, and the like. Although these analytical methods have good specificity, they all suffer from problems of more or less lengthy detection time, limited sensitivity, complicated preparation procedures or the need for large instruments. MPO-mediated production of HOCl is considered to be a biomarker related to oxidative stress-related injury, and the qualitative and quantitative determination of HOCl is of great significance to the research of disease progression. Currently, a variety of analytical methods for detecting HOCl have been established. Among them, fluorescent probes have been demonstrated to have many advantages such as high sensitivity, fast reaction time, simple operation, etc. However, there are some limitations to detecting HOCl using fluorescent probes: (1) the ultra-high sensitivity of the probe to active oxygen species, such as superoxide anion free radicals, hydroxyl free radicals and hydrogen peroxide, increases the risk of oxidation in air, and is not favorable for long-term storage; (2) it is difficult for the probe to quantitatively measure the actual concentration of HOCl because it is an intensity-based fluorescence output that can cause artifacts due to bleaching, focus variation, laser intensity variation, and probe concentration; (3) most fluorescent probes require complex, multi-step synthesis and suffer from significant drawbacks such as low yield, susceptibility to interference from probe concentrations, background fluorescence and insurmountable toxicity; (4) near Infrared (NIR) fluorescent probes, which are stable in the presence of HOCl, are lacking and are therefore very rare.

The introduction of nanotechnology can overcome many limitations of traditional probes, greatly improve the sensitivity and other performances of biosensors, promote the development of novel biosensors, and has important significance for early diagnosis of cancers and cardiovascular diseases. As a new type of nano material component, nucleic acid has the advantages of programmability, compatibility, good biocompatibility and the like. By introducing chemical modifications, a series of molecules with non-natural backbones or nucleobases can be synthesized. They have achieved significant success in probe design. In addition, nucleic acid framework materials (such as DNA tetrahedrons, DNA octahedrons, DNA icosahedrons, DNA cubes and the like) are also widely used for designing probe supports, so that the sensing performance of the probes is improved, the application range is widened, and the clinical feasibility of the DNA probes is promoted. In this work, we developed a three-dimensional DNA biosensor based on oxidative cleavage to rapidly and quantitatively detect HOCl and MPO in a "one-pot" manner.

Disclosure of Invention

In view of the above, the present invention aims to provide an isothermal nucleic acid amplification sensor for rapidly detecting hypochlorous acid and myeloperoxidase, so as to overcome the defects of the prior art, and analyze and quantitatively detect hypochlorous acid and myeloperoxidase by using a DNA framework structure-mediated enzyme-free catalytic reaction and combining electrophoresis and fluorescence technologies.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

an isothermal nucleic acid amplification sensor comprising three probes, probe P1, probe P2, and probe Lock-In:

the probe P1 is a three-dimensional structure probe formed by complementary base pairing of a DNA framework structure and a DNA hairpin 1, wherein the DNA hairpin 1 is modified with a fluorescent group or a fluorescence donor group;

the probe P2 is a three-dimensional structure probe formed by complementary base pairing of a DNA framework structure and a DNA hairpin 2, and the DNA hairpin 2 is modified with a fluorescence quenching group or a fluorescence receptor group;

the probe Lock-In is formed by two DNA single strands through base complementary pairing or one DNA hairpin single strand, the two DNA single strands are a Lock strand (Lock) and a trigger strand (In), wherein the Lock and the DNA hairpin single strands contain a phosphorothioate modified site, and the In and the DNA hairpin single strands can trigger DNA hairpin 1 In the probe P1 and DNA hairpin 2 In the probe P2 to perform DNA amplification reaction.

The nucleic acid nanosensor provided by the invention comprises three parts: a, probe 1(P1) which is respectively composed of a plurality of DNA single strands (S1, S2 … Sn and H1) to form a three-dimensional rigid nano structure; b, Probe 2(P2), three-dimensional rigid body composed of several DNA single strands (S1, S2 … Sn and H2)Sex nanostructures in which H1 and H2 constitute two units of an amplification reaction such as the Hybrid Chain Reaction (HCR); c, probe 3(Lock-In), a phosphorothioate modified nucleic acid constituting a Lock chain (Lock) or a DNA hairpin single chain, a priming chain (In) capable of initiating an amplification reaction such as a hybrid chain reaction, wherein In is initially locked by Lock and is incapable of initiating HCR, and H is catalyzed when a target Myeloperoxidase (MPO) is present₂O₂And chloride ion (Cl)^-) Hypochlorous acid (HOCl) is generated, the phosphorothioate chain is cleaved, and In is subsequently released, which triggers the amplification reaction of H1 and H2. The nucleic acid nanoprobe can detect hypochlorous acid and myeloperoxidase.

Preferably, the site of phosphorothioate modification is located in the middle of or near the middle of the DNA hairpin single strand.

One or more of Ferrocene (Ferrocene), Methylene Blue (Methylene Blue) and ruthenium (Ru) can be modified on the DNA hairpin 1 in the probe P1 and the DNA hairpin 2 in the probe P2, and the groups can generate electrochemical signals to realize the purpose of detection.

Preferably, the basic structure of probe P1 and probe P2 is a three-dimensional nanostructure selected from the group consisting of a Y-type structure, a DNA tetrahedron, a DNA hexahedron, a DNA octahedron, a DNA dodecahedron, a DNA hexadecahedron, or a DNA icosahedron; preferably, the number of bases on each side of the three-dimensional nanostructure of probe P1 and probe P2 is not less than 18 bp.

The three-dimensional structure is multidirectional, the reaction collision probability is increased, the reaction speed is greatly improved, and in addition, the three-dimensional nano structure can also increase the stability of the DNA hairpin in a complex environment and cannot be degraded by enzymes of a biological matrix. Similar effects can be obtained if other three-dimensional structures are used. Of course, one-dimensional or two-dimensional structures may also be used, but the effect is relatively poor compared to three-dimensional structures.

Preferably, the three-dimensional nanostructures of probe P1 and probe P2 are DNA tetrahedrons; preferably, the number of bases of the 4 main chain DNA single strands forming the tetrahedral structure is 79bp, and the sequences are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.

The DNA tetrahedron is usually composed of four single strands forming each face, and is stable when each side is 18bp, that is, the DNA tetrahedron is most commonly constructed by four single strands with the length of 54 bp. Also, since the DNA tetrahedron S1-S4 need to be complementary to

DNA hairpins

1 and 2, and usually both strands need to be complementary around 18 bases at 37 ℃ for stability, the length of S1-S4 constituting the tetrahedron is at least 72bp or more, and we prefer 79bp in view of the hairpin to avoid non-specific reaction with the tetrahedral backbone.

Preferably, the number of bases of the DNA hairpin 1 of the probe P1 and the DNA hairpin 2 of the probe P2 is not less than 43 bp; preferably, the number of bases of DNA hairpin 1 of probe P1 and DNA hairpin 2 of probe P2 is 67bp, and the sequences are shown as SEQ ID NO.7 and SEQ ID NO.8 respectively.

The temperature set by the conventional hybrid chain reaction is generally at 37 ℃, two hairpins are required to be in a silent state when no priming chain exists, the hairpins are required to be opened rapidly to react under the condition that the priming chain exists, the effective reaction length of the DNA hairpins is 43bp through research, and the base numbers of the

DNA hairpins

1 and 2 are not less than 61bp because the effective reaction length of the

DNA hairpins

1 and 2 and four main chains of a tetrahedron are not less than 18bp through hybridization reaction. The number of bases of DNA hairpin 1 on probe P1 and DNA hairpin 2 on probe P2 is preferably 67bp, considering that the hairpins are to avoid non-specific reactions with the tetrahedral backbone.

Preferably, In and the number of bases complementary to DNA hairpin 1 In probe P1 are not less than 22, In and the number of bases complementary to DNA hairpin 2 In probe P2 are not less than 22, and the number of bases complementary to DNA hairpin 1 In probe P1 and DNA hairpin 2 In probe P2 are not less than 21.

According to the above, the effective reaction length of the DNA hairpin is preferably 43bp, and at this time, when the sequence length of the priming strand is 22bp, the hairpin can be opened more quickly to react.

Preferably, the number of bases of In is not less than 22 bp; the base number of Lock is not less than 18bp, wherein, the number of the phosphorothioate modification sites is not less than 1; when the Lock-In occurs In a form of 2 DNA single strands through base complementary pairing, the number of bases complementary to the Lock and the In is not less than 18 bp; when the Lock-In is In the form of DNA hairpin single-chain, the number of complementary bases is not less than 18; preferably, the sequences of Lock and In are shown as SEQ ID NO.5 and SEQ ID NO.6, respectively.

According to the previous item, when the sequence length of the initiating chain is 22bp, the Lock needs to be complementary with the initiating chain In at 37 ℃, and the complementary base number is not less than 18bp, so that the initiating chain can be completely locked.

Preferably, the fluorescent group on DNA hairpin 1 is adjacent to the quencher group of DNA hairpin 2 after base-complementarity, and/or the fluorescent donor group on DNA hairpin 1 is adjacent to the fluorescent acceptor group of DNA hairpin 2 after base-complementarity; the fluorescent group is selected from JOE, HEX, VIC, ROX, CY3 or CY5, and the quenching group is selected from BHQ1, BHQ2 or BHQ 3; the fluorescence donor group is selected from FAM or CY3, and the fluorescence acceptor group is selected from TAMRA or CY 5; preferably, the fluorescent group is FAM and the quencher group is BHQ 1; preferably, the fluorescence donor group is CY3 and the quencher group is CY 5.

The object of the invention can also be achieved using different combinations of fluorescence. Two conditions need to be met for the fluorophore to undergo fluorescence energy resonance transfer: (1) the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor; (2) the distance between the fluorescence donor and the fluorescence acceptor is shorter and is not more than 10 nm. FAM/BHQ1 this pair of fluorescence/quenching combinations is most common in all fluorescent labels and is also relatively inexpensive. CY3/CY5 this is for fluorescence donor/acceptor groups because they are not easily affected by the acidity or basicity of the system and their fluorescence transfer efficiency is high.

Taking a tetrahedron as an example, the DNA hairpins on four vertexes of the probe P1 are modified with a fluorescent group in the middle, the DNA hairpins on four vertexes of the probe P2 are modified with a fluorescence quenching group at the 3' end; if the DNA hairpin of the probe P1 is modified with a fluorescence donor group in the middle, the 3' end of the DNA hairpin of the probe P2 is modified with a fluorescence acceptor group. When the end of the DNA is modified, the cost is the lowest, the synthesis is easy and the yield is high.

The second objective of the invention is to provide a method for preparing an isothermal nucleic acid amplification sensor, which comprises the following steps:

(a) design and Synthesis of probes P1 or P2: mixing a plurality of DNA single strands for assembling a DNA framework structure and the DNA hairpin 1 or the DNA hairpin 2 in a buffer solution according to a ratio, heating and denaturing at 85-100 ℃ for 5-10min, and then keeping at 37 ℃ or below for 1-30min to obtain a probe P1 or P2;

(b) designing and synthesizing a probe Lock-In: mixing 2 DNA single-chains Lock and In a buffer solution according to a proportion or mixing DNA hairpin single-chains In the buffer solution, heating and denaturing at 85-100 ℃ for 5-10min, and then slowly cooling to 37 ℃ or below to obtain a probe Lock-In.

Because the strand of nucleic acid is negatively charged, cations are added to neutralize the electronegativity of the strand of nucleic acid during complementation so as to complement the double strands, wherein sodium ions and magnesium ions are most commonly used. Preferably, in step (a), the buffer has a magnesium ion content of not less than 10 mM; more preferably, the buffer solution has a magnesium ion concentration of 50mM and is denatured by heating at 95 ℃ for 5 min;

in the step (b), the concentration of sodium chloride In the buffer solution is not less than 100mM or the content of magnesium ions is not less than 5mM, and the molar ratio of Lock to In is not less than 1: 1; preferably, Lock and In are In a molar concentration ratio of 1: 1.

The third purpose of the invention is to provide the application of the isothermal nucleic acid amplification sensor in biological detection, in particular the application in detecting hypochlorous acid and myeloperoxidase.

Compared with the prior art, the isothermal nucleic acid amplification sensor for rapidly detecting hypochlorous acid and myeloperoxidase has the following beneficial effects:

(1) the probe is prepared quickly and controllably: the construction of the probe takes only 5 minutes to complete. Compared with the existing method which usually needs several hours, the detection time does not exceed 30 minutes, and the detection efficiency of the sensor is greatly improved.

(2) One-pot detection: the cleavage reaction of the PS modified site does not need complex reaction conditions and operation, and only needs to place the probe at 37 ℃ and measure fluorescence after half an hour.

(3) Proportional quantitative detection: the proportional-type detection can prevent false positive signals, and the ratiometric detection also has the advantage of quantifying the analyte. The biosensor is not affected by the pH range, buffer type and matrix.

(4) Stable signal output and long term storage: the performance of the sensor changed little over 6 months, indicating that the biosensor has good stability even after long-term storage.

(5) Simplicity and safety: the biosensor does not involve complicated organic synthesis, easily degradable proteases, trained operators, expensive instruments and dedicated laboratory space, which enables reliable detection results to be obtained even in resource-limited environments. In addition, no reagent contains hazardous substances and is not user-friendly.

(6) Strong versatility: the design of the signal amplification nucleic acid circuit based on the hybridization chain reaction can be expanded to other signal amplification strategies with higher sensitivity.

Therefore, our work can be widely applied not only to the related research fields of biosensors and molecular diagnostics, but also to the commercial application.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a diagram of the nucleic acid nanosensor structure and detection mechanism of the invention, wherein FIG. 1a is a diagram showing hypochlorous acid cleaving a phosphorothioate-modified nucleic acid at specific sites; FIG. 1b is a tetrahedron-mediated hybridization chain reaction for detecting hypochlorous acid; FIG. 1c is a tetrahedron-mediated hybridization chain reaction for detecting MPO;

FIG. 2 is a diagram of the synthesis and characterization of the nucleic acid nanosensor structure of the invention, wherein FIG. 2a is a representation of polyacrylamide gel electrophoresis of HOCl-cleaved Lock-In at different concentrations; FIG. 2b is a representation of polyacrylamide gel electrophoresis of a conventional HCR reaction initiated by hypochlorous acid; FIG. 2c is a representation of polyacrylamide gel electrophoresis of probes P1 and P2; FIG. 2d is a diagram showing the agarose gel electrophoresis of starting TDN-HCR after hypochlorous acid is added;

FIG. 3 is a diagram showing the fluorescence result of hypochlorous acid detected by the nucleic acid nanosensor of the invention, wherein FIG. 3a is a diagram showing the detection of the fluorescence ratio of TDN-HCR to HOCl; FIG. 3b shows fluorescence spectra and FRET ratios (F) with different components added_A/F_D) Change, Line 1: p1+ P2+ Lock-In + HOCl, Line 2: p1+ P2+ Lock-In, Line 3: p1+ P2; FIG. 3c shows R/R after addition of different Lock sequences₀(ii) a change in (c); FIG. 3d is the fluorescence spectrum of the biosensor after the addition of HOCl (0-10. mu.M); FIG. 3e is a HOCl concentration dependent F_A/F_DVariation from HOCl (0-10. mu.M) concentration and inset is F in the range 0nM-200nM_A/F_DLinear relationship to HOCl concentration; FIG. 3F is F of biosensor vs. HOCl (500nM) and other analytes (5. mu.M)_A/F_DVariation, conditions: [ P1]＝[P2]＝50nM，[H1]＝[H2]＝200nM，[Lock-In]＝500nM，[HOCl]Reaction at 37 deg.c for 30min at 1 μ M, λ ex 548 nm;

FIG. 4 is a graph showing the results of fluorescence detection of MPO by the nucleic acid nanosensor of the invention, wherein FIG. 4a is a graph showing fluorescence spectra and FRET ratios (F) after addition of different components_A/F_D)，Line1：P1+P2+Lock-In+MPO+Cl^-+H₂O₂，Line2：P1+P2+Lock-In+MPO+Cl^-，Line3：P1+P2+Lock-In+MPO+H₂O₂，Line4：P1+P2+Lock-In+Cl^-+H₂O₂Line 5: p1+ P2+ Lock-In; FIG. 4b shows F after addition of MPO_A/F_DTime dependence of (d); FIG. 4c is a graph showing MPO concentration-dependent fluorescence spectrum and F in the range of 0 to 1. mu.g/mL_A/F_DA change in (c); FIG. 4d is a graph of F at 2-25ng/mL_A/F_DA linear relationship with the logarithm of the MPO concentration; FIG. 4e is F of biosensor versus MPO (50ng/mL) and other analytes (500ng/mL)_A/F_D(ii) a change; FIG. 4f anti-interference capability of the biosensor against common physiological substances; FIG. 4g is a graph of the effect of different concentrations of 4-ABAH on the chlorination activity of MPO; FIG. 4h is the long term stability of the biosensor for more than 6 months; FIGS. e-h are plots of 50ng/mL MPO concentration, conditions: [ P1]＝[P2]＝50nM，[H1]＝[H2]＝200nM，[Lock-In]＝200nM，[H₂O₂]＝[Cl^-]＝100μM，PB 60, reacting at 37 ℃ for 30 min;

FIG. 5 is a fluorescence photograph and a fluorescence intensity histogram of six samples with and without MPO, in which a MPO is added to PBS 7.4, as a result of detecting the fluorescence of MPO in an actual sample by the nucleic acid nanosensor of the invention; b, adding MPO into the plasma; adding MPO in serum; d, adding MPO in the cell lysate; e MPO added into saliva; f: PBS without MPO.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. 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. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention. Unless otherwise indicated, the methods referred to in the examples of the present invention are, unless otherwise indicated, conventional techniques and methods of the relevant art. Materials, reagents, equipment and the like used in examples are commercially available unless otherwise specified.

The nucleic acid sequences used in the examples of the present invention are shown in Table 1 below, and the oligonucleotides were synthesized by Shanghai Biotechnology Ltd and purified by high performance liquid chromatography before use. The fluorescence spectrum was measured by Hitachi RF-5301 fluorescence spectrometer (Hitachi, Japan). Gel electrophoresis results were obtained from a gel recording system (Viffo, Beijing, China). The 96-well plates were measured on a BioTek Synergy 4 microplate reader (BioTek Instruments inc., USA).

TABLE 1 nucleic acid sequences employed in the examples of the invention

Note: and if the base is followed by the base in the Lock, the base is modified by the phosphorothioate.

Example 1: synthesis of probes P1, P2 and Lock-In

The nucleic acid nanosensor of the invention comprises three parts: a, probe 1(P1), which is a tetrahedral rigid nanostructure composed of five DNA single strands (S1, S2, S3, S4 and H1); b, probe 2(P2), which is a tetrahedral rigid nanostructure composed of five DNA single strands (S1, S2, S3, S4 and H2), wherein H1 and H2 constitute two units of an enzyme-free amplification reaction such as Hybrid Chain Reaction (HCR); c, probe 3(Lock-In), phosphorothioate modified nucleic acid constituting a chain (Lock), a primer chain (In) capable of initiating a hybridization chain reaction, wherein In is initially locked by Lock, unable to initiate HCR, catalyzes H when the target myeloperoxidase is present₂O₂And chloride ion (Cl-) to generate hypochlorous acid (HOCl), the phosphorothioate chain is broken, and In is released, so that the enzyme-free amplification reaction of H1 and H2 is initiated.

(1) DNA single strands S1, S2, S3, S4, H1, H2, H1, H2, Lock and In (Table 1) were diluted to 20. mu.M with enzyme-free water and vortexed for 10 seconds to mix the single strands thoroughly. Subpackaging, storing at 4 deg.C for a short time, and storing at-20 deg.C for a long time. The obtained probe was used directly in the subsequent experiments without further isolation or purification.

(2) Synthesis of Probe P1: 1 μ M of S1, S2, S3 and S4 was mixed with 4 μ M H1 in Tris-HCl-Mg²⁺Buffer (20mM Tris-HCl, 50mM MgCl)₂pH 8.0), mixing, centrifuging, heating at 95 deg.C for 5min, immediately cooling on ice, and storing at 4 deg.C. The obtained probe was used directly in the subsequent experiments without further isolation or purification.

(3) Synthesis of Probe P2: 1 μ M of S1, S2, S3 and S4 was mixed with4 μ M H2 mix in Tris-HCl-Mg²⁺Buffer (20mM Tris-HCl, 50mM MgCl)₂pH 8.0), mixing, centrifuging, heating at 95 deg.C for 5min, immediately cooling on ice, and storing at 4 deg.C. The obtained probe was used directly in the subsequent experiments without further isolation or purification.

(4) Synthesis of a Probe Lock-In: the chain (Lock, 10. mu.M) and the initiating chain (In, 10. mu.M) In equal proportions are mixed In 1 XPBS (0.01M, pH 7.4) buffer, mixed evenly and centrifuged, heated at 95 ℃ for 5min, slowly cooled to 25 ℃, reacted for 30min, and stored at 4 ℃ for later use.

Example 2: electrophoretic characterization of the probes

(1) HOCl cleavage of Lock-In: fluorescently labeled Lock (2. mu.M) or fluorescently labeled Lock-In (2. mu.M) was incubated with different concentrations of hypochlorous acid In 1 XPBS buffer at 37 ℃ for 1 h. And (4) performing characterization on the Lock-In and the cleavage product by using 10% polyacrylamide gel electrophoresis. The electrophoresis separation is carried out for 80min at 4 ℃ and 120V. The gel was placed in a GelImage system at room temperature for imaging observation.

(2) HOCl triggers the classical HCR: h1, h2, 2 μ M each of In or Lock-In was incubated with varying concentrations of hypochlorous acid In 1 XPBS buffer at 37 ℃ for 4 h. The chain and cleavage products were characterized by 10% polyacrylamide gel electrophoresis. The electrophoresis separation is carried out for 80min at 4 ℃ and 120V. And (3) placing the gel into GelRed for dyeing for 15min at room temperature, and then placing the gel into a GelImage system for imaging observation.

(3) Construction of probes P1 and P2: adding 1 μ MS1, S2, S3, S4, 4 μ M H1 and H2 into 200 μ L PCR tube according to the precise proportion, and adding into Tris-HCl-Mg²⁺Buffer (20mM Tris-HCl, 50mM MgCl)₂pH 8.0) and then centrifuged. Heating at 95 deg.C for 5min, and immediately cooling on ice. Characterization was performed using 6% polyacrylamide gel electrophoresis. The electrophoresis separation is carried out for 80min at 4 ℃ and 120V. And (3) placing the gel into GelRed for dyeing for 15min at room temperature, and then placing the gel into a GelImage system for imaging observation.

(4) HOCl initiated TDN-HCR: 500nM P1 and P2, 2. mu.M Lock-In and In were incubated with varying concentrations of hypochlorous acid In 1 XPBS buffer at 37 ℃ for 30 min. The assembled product was characterized by electrophoresis on a 2% agarose gel. The electrophoresis separation is carried out for 30min at the temperature of 4 ℃ and the pressure of 80V. The Gel is stained by GelRed in advance, and after the separation operation is finished, the Gel is placed into a Gel Image system for imaging observation.

Example 3: probe for detecting hypochlorous acid in vitro

(I) Synthesis of Probe

(1) Synthesis of fluorescently labeled Probe P1: mu.M of S1, S2, S3 and S4 was mixed with 4. mu.MH 1(Cy3 marker) in Tris-HCl-Mg²⁺Buffer (20mM Tris-HCl, 50mM MgCl)₂pH 8.0), mixing, centrifuging, heating at 95 deg.C for 5min, immediately cooling on ice, and storing at 4 deg.C. The obtained probe was used directly in the subsequent experiments without further isolation or purification.

(2) Synthesis of fluorescently labeled Probe P2: mu.M of S1, S2, S3 and S4 was mixed with 4. mu. M H2 (labeled with Cy 5) in Tris-HCl-Mg²⁺Buffer (20mM Tris-HCl, 50mM MgCl)₂pH 8.0), mixing, centrifuging, heating at 95 deg.C for 5min, immediately cooling on ice, and storing at 4 deg.C. The obtained probe was used directly in the subsequent experiments without further isolation or purification.

(II) in vitro identification and results

Fluorescence spectrum of hypochlorous acid response: 50nM P1, 50nM P2 and 500nM Lock-In were mixed well In 100. mu.L PBS buffer (pH 7.4) and incubated with different concentrations of HOCl (0 to 10. mu.M) for 30min at 37 ℃. The fluorescence spectrum of the mixture at 550 to 800nm was then collected under excitation at 548 nm. Wherein, F_AAnd F_DFluorescence of Cy5 at 665nm and Cy3 at 561nm, respectively. All experiments were repeated at least three times.

And (3) dynamic study: 50nM P1, 50nM P2 and 500nM Lock-In were mixed well In 100. mu.L PBS buffer (pH 7.4), temperature controlled at 37 ℃ and initial fluorescence intensity measurements at 561nM and 665nM were started. Spectra were taken at 561nm and 665nm channels every 1min after adding HOCl. At each time point F_A/F_DPlotted against time.

Specific experiments: NaOCl (ε)_292nm＝350M^-1cm^-1) Aqueous solution as HOCl donor. H₂O₂(ε_240nm＝43.6M^- ¹cm^-1) Aqueous solution as H₂O₂A donor. Xanthine/xanthine oxidase as superoxide (O)₂ ^-) A donor. By adding an excess of H to₂O₂Adding Fe to the solution²⁺OH is produced. TBHP stock solution (10mM) was prepared by adding 998.8. mu.L of H₂O to which was added 1.2. mu.L of 70% TBHP. Stock NO (10mM) was prepared by dissolving 2.98mg sodium nitroprusside in 1mL of H₂Obtained in O. To the probe (50nM P1, 50nM P2 and 500nM Lock-In) was added ROS/RNS generator In 100 μ L PBS buffer (pH 7.4) at a final concentration of 10 μ M interferent and 1 μ M HOCl. The samples were incubated at 37 ℃ for 30min and the fluorescence spectra recorded.

Example 4: probe for in vitro detection of MPO

(I) Synthesis of Probe

The method is the same as that of example 3 (A)

(II) in vitro identification and results

Fluorescence spectrum of MPO response: MPO solutions were prepared by dissolving MPO in 1mL of water (20% glycerol). The solution was stored at 4 ℃ and used within 2 weeks. 50nM P1, 50nM P2, and 200nM Lock-In were mixed well In 100. mu.L PBS buffer (pH 6.0) and incubated with different concentrations of MPO (0 to 1. mu.g/mL) at 37 ℃ for 30 min. The fluorescence spectrum of the mixture at 550 to 800nm was then collected under excitation at 548 nm. Wherein, F_AAnd F_DFluorescence of Cy5 at 665nm and Cy3 at 561nm, respectively. All experiments were repeated at least three times.

And (3) dynamic study: 50nM P1, 50nM P2 and 200nM Lock-In were mixed well In 100. mu.L PBS buffer (pH 6.0), temperature controlled at 37 ℃ and initial fluorescence intensity measurements at 561nM and 665nM were started. MPO was added and spectra were taken at 561nm and 665nm channels every 1 min. At each time point F_A/F_DPlotted against time.

To explore the effect of halogen ions on the system, MPO standard solution should be centrifuged 3 times using 10k ultrafilter tubes (10k, Amicon Ultra-0.5) to remove chloride before cleavage reaction.

An inhibitor of MPO (4-ABAH) was diluted in DMSO. In this experiment, 4-ABAH was added directly to MPO (50ng/mL) solution and incubated at 37 ℃ for 30min prior to lysis.

Example 5: probe for detecting MPO in complex matrix

(I) Synthesis of Probe

The method is the same as that of example 3 (A)

(II) recovery test

(1) Serum/plasma: human serum and plasma were purchased from commercial sources and used after 100-fold dilution with PBS. Recovery tests were carried out by adding different amounts of MPO to diluted human serum and plasma samples.

(2) Cell lysis solution: mouse mononuclear macrophage RAW264.7 was obtained from chinese academy of sciences type culture bank cell bank (shanghai, china) and cultured in dmem (gibco) medium containing 10% FBS and 1% penicillin-streptomycin (PS, 10000IU penicillin and 10000 μ g/mL streptomycin). Cells were passaged every 3 days, and after 30 passages, culture was resumed from the stock. Cell lysates were prepared by mixing cells with mammalian cell lysis buffer (50mM Tris-HCl, 150mM NaCl, 0.1% SDS, 0.5% deoxycholic acid and protease inhibitor) followed by centrifugation to remove nuclear DNA and cell membrane debris. Recovery tests were carried out by adding different amounts of MPO to 100-fold diluted cell lysate samples.

(3) Saliva: unstimulated whole human saliva samples were collected in the early morning prior to any food/beverage consumption. The samples were centrifuged at 10,000g for 30min and the clear supernatant was transferred to a microcentrifuge tube and stored in a freezer at-20 ℃ when not in use. Recovery tests were carried out by adding different amounts of MPO to 100-fold diluted human saliva samples.

Example 6: portable device for detecting MPO activity

(I) Synthesis of Probe

(1) Synthesis of FAM/BHQ 1-labeled Probe P1: mu.M of S1, S2, S3 and S4 and 4. mu. M H1(FAM/BHQ1 marker) were mixed in Tris-HCl-Mg²⁺Buffer (20mM Tris-HCl, 50mM MgCl)₂pH 8.0), mixing, centrifuging at 95 deg.CHeating for 5min, immediately cooling on ice, and storing at 4 deg.C. The obtained probe was used directly in the subsequent experiments without further isolation or purification.

(2) Synthesis of Probe P2: 1 μ M of S1, S2, S3 and S4 was mixed with 4 μ M H2 in Tris-HCl-Mg²⁺Buffer (20mM Tris-HCl, 50mM MgCl)₂pH 8.0), mixing, centrifuging, heating at 95 deg.C for 5min, immediately cooling on ice, and storing at 4 deg.C. The obtained probe was used directly in the subsequent experiments without further isolation or purification.

(II) construction of Portable devices

The PCR tube was placed in a home-made room, excited with uv light, and the fluorescence image captured with a smartphone. For semi-quantitative tests, the results can be read directly with the naked eye. All experiments were performed in triplicate to ensure reproducibility. The fluorescence image shot by the camera of the smartphone is transmitted to a computer and further analyzed by using image processing software ImageJ. By selecting the entire area of each detection zone, an average green channel value for each detection zone is obtained. The average green channel value of the blank sample was used as background and subtracted from the value at the detection zone.

Comparative example:

from tables 2 and 3, it can be seen that the isothermal nucleic acid amplification sensor of the present invention has the lowest detection limit known to us when detecting hypochlorous acid, compared to other methods based on colorimetric or fluorescent detection. When the isothermal nucleic acid amplification sensor is used for detecting MPO, the effect is superior to or can be comparable with that of a reported colorimetric method, a fluorescence method and an electrochemical detection method. Although higher LOD than immunoassays, the greatly simplified procedure and significantly reduced time make our method more suitable for practical applications.

TABLE 2 comparison of different fluorescence methods for detecting HOCl

TABLE 3 comparison of different analytical methods for the detection of MPO

The structure and performance of the isothermal nucleic acid amplification sensor prepared according to the present invention will be described in detail with reference to the accompanying drawings

Experimental example 1: TDN-HCR-based working mechanism analysis of HOCl sensing strategy

When one of the non-bridging oxygens of the nucleic acid structure is modified by phosphorothioates (ps), cleavage occurs at a specific site by addition of an appropriate amount of HOCl (fig. 1 a). Since the phosphorothioate modified nucleic acid only slightly perturbs the structure of the DNA, base pairing is not affected, and the programmability of the DNA is retained.

In this work, we developed a TDN-HCR nanosensor to detect HOCl (FIG. 1 b). The Hybrid Chain Reaction (HCR) is a highly convenient and multifunctional enzyme-free isothermal amplification technique for nucleic acids. In the conventional HCR, two hairpin probes (h1 and h2) are alternately opened under the trigger of an initiating strand (In), and a grown linear double-stranded product is assembled and accompanied by signal output such as Fluorescence Resonance Energy Transfer (FRET). However, the conventional enzyme-free reaction depends on random collision and interaction of nucleic acid molecules in a solution, and has the problems of weak biological stability, slow reaction kinetics, low hybridization efficiency and the like. To accelerate the reaction kinetics of HCR, we utilized a Tetrahedral DNA Nanostructure (TDN) mediated hyperbranched HCR strategy. H1 and h2 are fixed on the four vertices of the TDN respectively. To complement the overhangs of the four strands of TDN, T was extended at h1 and h2₂₂G₂Hairpins H1 and H2 were designed. Subsequently, by connecting H1 and H2 to the four vertices of TDN, two sets of TDN hairpin units (P1 and P2) can be obtained. After the initiation chain (In) is added, the H1 hairpin In P1 is opened, and the released sequence can further open the H2 hairpin In P2 to form an H1/H2 complex, and finallyFinal assembly forms hyperbranched nucleic acid structures. Unlike conventional HCR, we can increase the effective collision probability and increase the local concentration by introducing DNA hairpins at the four apex angles of TDN, thus greatly accelerating the reaction kinetics. Next, we designed a chain (Lock) containing phosphorothioate modification based on the sequence characteristics of the initiating chain, and In the absence of HOCl, the chain formed a complex (Lock-In) with the initiating chain, making the HCR reaction impossible. When hypochlorous acid was added at various concentrations, the site of phosphorothioate In the chain was cleaved, the chain structure was unstable, and the initiating chain was released from the complex (Lock-In), thereby initiating the HCR reaction to detect hypochlorous acid (FIG. 1 b). Under physiological conditions, MPO mediates H₂O₂For chloride ion (Cl)^-) To produce HOCl. Therefore, our proposed strategy can be easily extended to monitoring MPO activity (FIG. 1 c).

Experimental example 2: electrophoretic analysis

The feasibility of the sensor is preliminarily evaluated by polyacrylamide gel electrophoresis (PAGE) and Agarose Gel Electrophoresis (AGE), and the design scheme is preliminarily evaluated. (1) Block-In was cleaved by HOCl: as shown In FIG. 2a, the hybridization of Lock and In forms a stable Lock-In double strand, which has slower mobility than Lock. Meanwhile, when Lock-In was treated with HOCl, a new band corresponding to the fluorescently labeled cleaved fragment of Lock appeared, and the degree of cleavage correlated with HOCl concentration, indicating that the Lock strand was cleaved and released from the Lock-In double strand. (2) HOCl initiates the traditional HCR reaction: as shown in FIG. 2b, h1, h2, and h1+ h2 show one DNA band (

lanes

2, 7, 8), respectively. After addition of In, a long-gapped double-stranded DNA band similar to the alternating copolymer was clearly visible on the gel (lane 1). When In was substituted by Lock-In, the HCR process was inhibited (lane 6). The HCR process can be automated again when In is released from Lock by HOCl. From the results in lanes 3-5, it is clear that the presence of HOCl is essential for initiating the HCR process. (3) HOCl-initiated TDN-HCR: with the successive addition of oligonucleotides S1, S2, S3, S4 and H1 or H2, a significant drop in electrophoretic mobility was observed, indicating that P1 and P2 have been successfully prepared (fig. 2 c). P1 and P2 can be substituted for h1 and h2, respectively, for HOCl-initiated TDN-HCR. The P1/P2 and P1/P2/Lock-In mixtures gave similar DNA bands (

lanes

1 and 2, FIG. 2d), indicating that Lock-In does not spontaneously initiate TDN-HCR. However, in the presence of HOCl, a large size HCR product was formed (lanes 3-5, FIG. 2d), which failed to migrate from the loading well, indicating that HOCl successfully initiated TDN-HCR. It should be noted that even 0.5. mu.M HOCl is effective in activating TDN-HCR, which means that the HOCl-detecting ability of TDN-HCR is greatly improved as compared with conventional HCR.

Experimental example 3: FRET-based HOCl ratio-based detection

The fluorescence signal output mode based on FRET is obviously superior to the common fluorescence/quenching system because the false positive signal caused by probe degradation, concentration fluctuation and excitation light intensity change can be effectively prevented. In order to realize FRET detection of HOCl in the solution, a fluorophore donor Cy3 and a fluorophore acceptor Cy5 were modified at appropriate positions of H1 and H2, respectively. After HOCl successfully triggers TDN-HCR, FRET between them will change from "off" to "on" state (fig. 3 a). To confirm the feasibility of our proposed HOCl sensing strategy, a fluorescence analysis was first performed. As shown In FIG. 2b, the P1/P2 and P1/P2/Lock-In mixtures gave the same fluorescence spectra (Line2, 3). When HOCl was added to the P1/P2/Lock-In mixture, TDN-HCR was successfully initiated, resulting In a substantial decrease In Cy3 fluorescence and a significant increase In Cy5 fluorescence (Line 1). FRET ratio (F) calculated from fluorescence spectrum_A/F_D) In (1), F containing HOCl_A/F_DAbout 12.7 times higher (F) than the control without HOCl_ARefers to the emission signal value of Cy5 at 665nm after being excited at 548 nm; f_DIs the value of the emission signal at 561nm after Cy3 was excited at 548 nm).

After proving the feasibility of ratio detection for HOCl, we optimized the sensor design and some important experimental conditions in order to achieve the best detection effect. First, we select the best Lock (fig. 3 c). An ideal Lock should hybridize to In and remain silent In the absence of HOCl, but can be cleaved rapidly by HOCl to release In, thereby initiating a subsequent HCR reaction. Therefore, we carefully choose the length of Lock andnumber and position of PS modifications in Lock. We compared four Lock chains of the same length but with different numbers of PS modifications (Lock0, Lock1, Lock2 and Lock3, where the PS modification sites are 0, 1, 2 and 3, respectively). Due to the lack of PS modification, the Lock0-In duplex was not reactive to HOCl and did not initiate HOCl-dependent TDN-HCR. In contrast, all of Lock1-In, Lock2-In, and Lock3-In reacted to HOCl to yield F_A/F_DIs greatly increased. However, when the number of modification points is two or more, the background signal increases sharply. The Tm for each PS site is reported to drop on average by 0.65 ℃ compared to the unmodified duplex. The effect of PS modification on double strand instability can be overcome by increasing the length of the double strand. When the length of Lock is extended and the PS modification site is gradually increased, like Lock4-6, HCR remains silent in the absence of HOCl. Of these, Lock5-In and Lock6-In can be rapidly cleaved by HOCl to release In, triggering the subsequent HCR process, while Lock4 cannot. Thus, the PS modification site should be designed near the middle of the lock. This allows maximum In release after cleavage of the duplex by HOCl.

Then, with R/R₀Is a standard (R and R)₀Respectively, F containing HOCl and F not containing HOCl_A/F_D) The operating conditions for HOCl detection including the concentration and ratio of DNA probes (P1, P2 and Lock-In), buffer type and pH, reaction time were optimized. Wherein, the concentration, proportion and reaction time of the probe have obvious influence on the reaction, and the type and pH value of the buffer solution have little influence. Preferably, 50nM P1, 50nM P2, 500nM Lock-In, 30min reaction time was chosen for the detection of HOCl In the next experiment.

Under optimized conditions, the sensitivity of the HOCl sensing platform was analyzed. F increasing from 0 to 10. mu.M of HOCl concentration_A/F_DThe value gradually increases (fig. 3 d). F_A/F_DValues were linearly related to HOCl concentration in the range of 2.5nM to 200nM (fig. 3 e). The regression equation is that Y is 0.0089X +0.2025 (wherein Y and X respectively represent F_A/F_DAnd HOCl concentration), the limit of detection (LOD) was estimated according to the 3 σ/S rule (σ represents the standard deviation of the blank (N ═ 10), S is the slope of the calibration curve), as0.8 nM. Notably, the LOD of this biosensor is the lowest known to us compared to other methods based on colorimetric or fluorescent detection (table 2). It is noteworthy that although the sensing process includes a HOCl triggered Lock-In cleavage and released In triggered TDN-HCR, the operation of these two steps can be combined, allowing the entire HOCl detection to be done In a "one pot" mode, which greatly simplifies operation.

It is well known that biosensors having high selectivity and interference immunity are advantageous for accurate detection of HOCl due to the presence of large amounts of reactive oxygen and reducing species in biological systems. We further investigated the selectivity of the biosensor for HOCl and other potentially interfering analytes. As shown in FIG. 3F, only HOCl contributed to the fluorescence intensity ratio (F)_A/F_D) While other interfering analytes show negligible F even at 10-fold higher concentrations_A/F_DAnd (4) changing. These results demonstrate that our biosensor can achieve highly selective detection of HOCl in a complex biological system.

Experimental example 4: FRET-based MPO ratiometric detection

The addition of MPO in the presence of Cl-and H2O2 greatly increased the FRET signal of the sensing system, demonstrating the feasibility of our method for the detection of MPO. In contrast, little change in FRET signal was observed for sensing systems lacking Cl-, H2O2, or MPO. This result strongly supports the detection mechanism we propose, that is, only in the presence of MPO, the oxidation of Cl-catalyzed by MPO to generate HOCl, and then the initiation of TDN-HCR leads to an increase in FERT signal output (FIG. 3 a). In order to obtain the best MPO detection effect, some key experimental conditions are optimized by taking R/R0 as a standard, including reaction temperature and Cl^-、H₂O₂And the concentration of Lock-In, buffer type and pH, and reaction time. Preferably, we selected 200nM Lock-In, 100. mu.M chloride, 100. mu.M H at 37 ℃₂O₂MPO was detected by using PB buffer solution (pH 6.0) and a reaction time of 30min (FIG. 3 b).

Under optimized conditions, the MPO detection performance of the method is evaluated. As shown in FIG. 3c, the FRET signal is closely related to the MPO concentration. F_A/F_DThe values show a sigmoidal curve with MPO over a concentration range of 0 to 1000 ng/mL. In the range of 2 to 25ng/mL, F_A/F_DIs linearly related to the logarithm of the MPO concentration (R)²0.9859) (fig. 3 d). The regression equation is that Y is 3.682X-0.4307, wherein Y and X respectively represent F_A/F_DRatio and logarithm of MPO concentration. The LOD is about 3.75ng/mL, which is superior to or comparable to the reported colorimetric, fluorescent, and electrochemical detection methods. Although higher LOD than the immunoassay (table 3), the greatly simplified procedure and significantly reduced time made our method more suitable for practical applications.

Then, the specificity of the MPO assay was investigated by assaying several other common enzymes (horseradish peroxidase, catalase, xanthine oxidase, glucose oxidase and glutathione reductase), proteins (bovine serum albumin) and various biologically relevant molecules such as metal ions, anions, redox substances, amino acids and glucose. As shown in fig. 3e and 3f, none of the tested biomolecules and ions gave a positive FRET signal compared to the blank.

Elevated MPO concentrations and activities are associated with a number of pathological factors that contribute to the development and progression of inflammatory events. Recent studies have shown that MPO can serve as an important biological diagnostic marker and therapeutic target. Therefore, screening for active compounds that are effective in inhibiting MPO activity, thereby attenuating the inflammatory response, is the focus of current research. To evaluate the potential of biosensors in screening for MPO inhibitors, we used 4-aminobenzoic acid hydrazide (4-ABAH) as a model, a drug widely used for MPO irreversible inhibitors. As shown in FIG. 3g, F_A/F_DThe ratio decreased with increasing concentration of 4-ABAH (0 to 15. mu.M). Calculated, half maximal Inhibitory Concentration (IC)₅₀) At 0.77 μ M, similar to the recent study report. This result suggests that the biosensor proposed by us can be used to screen MPO inhibitors and evaluate their inhibitory activity, showing great potential in the development of anti-inflammatory drugs.

A stable and reliable biosensor is crucial for the development of diagnostic kits. We stored the proposed sensing system at 4 ℃ for various periods of time and tracked its MPO detection capacity for 6 months. As shown in FIG. 3h, the background value of the sensing system hardly changed from the beginning to 6 months later, while the MPO sensing performance was little changed, indicating that the biosensor had good stability even after long-term storage.

Experimental example 5: determination of MPO Activity in actual samples

The biosensor is further used to detect MPO activity in an authentic sample. Taking plasma as an example, first, the effect of plasma dilution rate on MPO detection was tested. The same concentration of HOCl or MPO was added to samples of different dilutions, and it was found that approximately 100-fold dilution of plasma had no significant effect on the detection of MPO. However, in 10-fold dilutions of plasma, MPO could not be detected by adding more H₂O₂The reducing species in the sensing system cannot be neutralized and neutralized. Next, different concentrations of MPO were added to 100-fold diluted plasma samples, and the recovery was calculated by comparing the measured MPO activity and the added amount. For comparison, the MPO addition amount in the real sample was also determined using a commercial test kit. The recovery of MPO in plasma determined by our method was 95.6% to 102.3% with a relative standard deviation of 2.9% to 14.2%, which is consistent with the results of the commercial kit (table 4). This shows that our proposed method has good precision and high accuracy. Finally, our biosensor was also applied to three other real samples: serum, cell lysate and saliva (table 5). The results show that the biosensor can be used for quantitative determination of MPO in complex biological samples.

TABLE 4 recovery of MPO in plasma and comparison with commercial kits

TABLE 5 recovery of MPO in serum, cell lysates and saliva

Experimental example 6: portability of biosensor

The abnormal MPO causes the individual to have different susceptibility to some diseases, and is closely related to the occurrence and development of a plurality of human diseases, so that the rapid detection of the MPO is more and more concerned by scholars at home and abroad. Our biosensor can also be designed to be combined with a low-cost portable detection device to realize Point-of-care testing (POCT) of MPO. Therefore, we used a pair of fluorescence/quencher (FAM/BHQ1) to label H1. Due to the close contact between the fluorescent group in the hairpin structure and the quencher, the fluorescence of FAM is quenched. MPO can trigger TDN-HCR between P1 and P2, resulting in the opening of hairpin structure, thereby restoring the fluorescence of FAM. Four MPO positive authentic samples fluoresced brightly green by illuminating the sensing system with a hand-held UV lamp (FIG. 4). The detection result can be judged by naked eyes or a smart phone, and the fluorescence intensity can be semi-quantitatively graded. That is, the method proposed by us can be used to develop a portable MPO detection kit, and can be used even in resource-deficient or remote areas.

The nucleic acid sensor of the present invention utilizes a hybrid chain reaction for amplification, which is an enzyme-free amplification reaction, and the experimental purpose of the present invention can also be achieved by changing the nucleic acid sequence.

In the Lock-In the nucleic acid sensor, the number of bases of Lock can also reach the target by prolonging or increasing the phosphorothioate modification sites.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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Claims

1. An isothermal nucleic acid amplification sensor, comprising three probes, namely a probe P1, a probe P2 and a probe Lock-In, characterized In that:

2. The isothermal nucleic acid amplification sensor of claim 1, wherein: the basic structures of the probe P1 and the probe P2 are three-dimensional nanostructures selected from Y-shaped structures, DNA tetrahedrons, DNA hexahedrons, DNA octahedrons, DNA dodecahedrons, DNA hexadecahedrons or DNA icosahedrons; preferably, the number of bases on each side of the three-dimensional nanostructure of probe P1 and probe P2 is not less than 18 bp.

3. The isothermal nucleic acid amplification sensor of claim 2, wherein: the three-dimensional nano structures of the probe P1 and the probe P2 are DNA tetrahedrons; preferably, the number of bases of the 4 main chain DNA single strands forming the tetrahedral structure is 79bp, and the sequences are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.

4. The isothermal nucleic acid amplification sensor of claim 1, wherein: the base numbers of the DNA hairpin 1 of the probe P1 and the DNA hairpin 2 of the probe P2 are not less than 43 bp; preferably, the number of bases of the DNA hairpin 1 of the probe P1 and the DNA hairpin 2 of the probe P2 is 67bp, and the sequences are shown as SEQ ID NO.7 and SEQ ID NO.8 respectively.

5. The isothermal nucleic acid amplification sensor of claim 1, wherein: the number of bases of In complementary to the DNA hairpin 1 In the probe P1 is not less than 22, the number of bases of In complementary to the DNA hairpin 2 In the probe P2 is not less than 22, and the number of bases of the DNA hairpin 1 In the probe P1 complementary to the DNA hairpin 2 In the probe P2 is not less than 21.

6. The isothermal nucleic acid amplification sensor of claim 1, wherein: the base number of In is not less than 22 bp; the base number of Lock is not less than 18bp, wherein, the number of the phosphorothioate modification sites is not less than 1; when the Lock-In occurs In a form of 2 DNA single strands through base complementary pairing, the number of bases complementary to the Lock and the In is not less than 18 bp; when the Lock-In is In the form of DNA hairpin single-chain, the number of complementary bases is not less than 18; preferably, the sequences of Lock and In are shown In SEQ ID NO.5 and SEQ ID NO.6, respectively.

7. The isothermal nucleic acid amplification sensor of claim 1, wherein: the fluorescent group on the DNA hairpin 1 is adjacent to the quenching group of the DNA hairpin 2 after the base is complementary, and/or the fluorescent donor group on the DNA hairpin 1 is adjacent to the fluorescent acceptor group of the DNA hairpin 2 after the base is complementary; the fluorescent group is selected from JOE, HEX, VIC, ROX, CY3 or CY5, and the quenching group is selected from BHQ1, BHQ2 or BHQ 3; the fluorescence donor group is selected from FAM or CY3, and the fluorescence acceptor group is selected from TAMRA or CY 5; preferably, the fluorescent group is FAM and the quencher group is BHQ 1; preferably, the fluorescence donor group is CY3 and the quencher group is CY 5.

8. The method for preparing an isothermal nucleic acid amplification sensor according to any one of claims 1 to 7, wherein: the method comprises the following steps:

9. Use of the isothermal nucleic acid amplification sensor of any one of claims 1-8 in biological detection, in particular in the detection of hypochlorous acid and myeloperoxidase.