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

An isothermal nucleic acid amplification and sensor technology, applied in the field of nanomaterial synthesis and molecular detection, can solve the problems of unfavorable long-term storage, background fluorescence, difficult quantitative measurement, etc., to prevent false positive signals, improve detection efficiency, and stabilize long-term storage Effect

Pending Publication Date: 2021-12-03
NANKAI UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

However, the use of fluorescent probes to detect HOCl has some limitations: (1) The probe’s ultra-high sensitivity to reactive oxygen species, such as superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide, increases Risk, not conducive to 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, which will cause artifacts due to bleaching, focus changes, laser intensity changes, and probe concentration; ( 3) Most fluorescent probes require complicated multi-step synthesis, and have some obvious disadvantages, such as low yield, susceptibility to probe concentration interference, background fluorescence, and insurmountable toxicity; (4) lack of stability in the presence of HOCl near-infrared (NIR) fluorescent probes, so near-infrared fluorescent probes are very rare

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0053] Embodiment 1: synthetic probe P1, P2 and Lock-In

[0054] The nucleic acid nanosensor of the present invention comprises three parts: a, probe 1 (P1), respectively constitute tetrahedral rigid nanostructure by five kinds of DNA single strands (S1, S2, S3, S4 and H1); b, probe 2 (P2), respectively composed of five DNA single strands (S1, S2, S3, S4 and H2) to form a tetrahedral rigid nanostructure, wherein H1 and H2 constitute two enzyme-free amplification reactions such as hybridization chain reaction (HCR). Unit; c, probe 3 (Lock-In), phosphorothioate-modified nucleic acid constitutes a lock chain (Lock), which can initiate a chain reaction of hybridization (In), wherein In is initially locked by Lock and cannot initiate HCR, When target myeloperoxidase is present, it catalyzes H 2 o 2 And chloride ions (Cl-) to produce hypochlorous acid (HOCl), the phosphorothioate chain breaks, and then releases In, which triggers H1 and H2 to undergo enzyme-free amplification reac...

Embodiment 2

[0059] Embodiment 2: Electrophoretic characterization of probes

[0060] (1) HOCl cleavage of Lock-In: fluorescently labeled Lock (2 μM) or fluorescently labeled Lock-In (2 μM) was incubated with different concentrations of hypochlorous acid in 1×PBS buffer at 37° C. for 1 h. The Lock-In and cleavage products were characterized by 10% polyacrylamide gel electrophoresis. Under the conditions of 4°C and 120V, electrophoresis separation was carried out for 80 minutes. Place the gel into the GelImage system at room temperature for imaging observation.

[0061] (2) HOCl triggers classical HCR: 2 μM each of h1, h2, In or Lock-In was incubated with different concentrations of hypochlorous acid in 1×PBS buffer at 37° C. for 4 h. Use 10% polyacrylamide gel electrophoresis to characterize chains and cleavage products. Under the conditions of 4°C and 120V, electrophoresis separation was carried out for 80 minutes. After staining the gel in GelRed for 15 min at room temperature, put t...

Embodiment 3

[0064] Embodiment 3: Probe detects hypochlorous acid in vitro

[0065] (1) Synthetic probes

[0066] (1) Synthesis of fluorescently labeled probe P1: Mix 1 μM of S1, S2, S3 and S4 with 4 μM H1 (Cy3 labeled) in Tris-HCl-Mg 2+ Buffer (20mM Tris-HCl, 50mM MgCl 2 , pH=8.0), mixed and centrifuged, heated at 95°C for 5min, immediately cooled on ice cubes, and stored at 4°C until use. The obtained probes were used directly in subsequent experiments without further isolation or purification.

[0067] (2) Synthesis of fluorescently labeled probe P2: Mix 1 μM of S1, S2, S3 and S4 with 4 μM H2 (Cy5 labeled) in Tris-HCl-Mg 2+ Buffer (20mM Tris-HCl, 50mM MgCl 2 , pH=8.0), mixed and centrifuged, heated at 95°C for 5min, immediately cooled on ice cubes, and stored at 4°C until use. The obtained probes were used directly in subsequent experiments without further isolation or purification.

[0068] (2) In vitro identification and results

[0069] Fluorescence spectrum in response to hyp...

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Abstract

The invention provides an isothermal nucleic acid amplification sensor for rapidly detecting hypochlorous acid and myeloperoxidase, and the nucleic acid nano sensor comprises three parts: a, a probe 1 (P1), the probe 1 is a three-dimensional rigid nano structure formed by a plurality of DNA single chains (S1, S2... Sn and H1); b, a probe 2 (P2), the probe 2 is of a three-dimensional rigid nanostructure formed by a plurality of DNA single chains (S1, S2... Sn and H2), and H1 and H2 form two units of an amplification reaction such as a hybridization chain reaction (HCR); and c, a probe 3 (Lock-In), thiophosphoric acid modified nucleic acid which forms a lock chain (Lock) and an initiation chain (In) capable of initiating a hybridization chain reaction, In is locked by the Lock initially and cannot initiate HCR, and when a target object myeloperoxidase (MPO) exists, H2O2 and chloride ions (Cl-) are catalyzed to generate hypochlorous acid (HOCl), a thiophosphoric acid chain is broken, In is released, and the H1 and the H2 are initiated to generate an amplification reaction. The nucleic acid nanoprobe provided by the invention can be used for detecting the hypochlorous acid and the myeloperoxidase.

Description

technical field [0001] The invention relates to the technical field of nanomaterial synthesis and molecular detection, in particular to an isothermal nucleic acid amplification sensor for rapid detection of hypochlorous acid and myeloperoxidase mediated by DNA nanostructures. Background technique [0002] The pathological features of diseases are usually accompanied by the upregulation of certain biomarkers, such as ions, nucleic acids, small molecules and proteins. Detection tools with high sensitivity and strong compatibility are of great significance for the accurate diagnosis of diseases. However, the sensitivity, accuracy, and biosafety of probes remain challenges, which motivate researchers to explore more applicable materials and platforms to address these issues. Myeloperoxidase (myeloperoxidase, MPO), as a heme protease, mainly comes from polymorphonuclear neutrophils, and also exists in monocytes and macrophages. When the body is stimulated by inflammation and ph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825C12Q1/6806C12Q1/6851C12N15/11
CPCC12Q1/6825C12Q1/6806C12Q1/6851C12Q2525/301C12Q2525/113C12Q2525/00C12Q2527/125C12Q2563/107
Inventor 孔德明汪晶马嘉懿王东霞刘博井潇陈郸烨
Owner NANKAI UNIV
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