Method for detecting chain B of ricin in real time by using dual amplification strategy of catalytic hairpin assembly mediated lipidosome encoding magnetic beads

A ricin, double-amplification technology, applied in biochemical equipment and methods, material analysis by observing the impact on chemical indicators, microbial determination/inspection, etc., can solve the method of time-consuming, high immunogenicity, Complicated operation and other problems, to achieve the effect of convenient operation, high sensitivity and long time consumption

Active Publication Date: 2020-07-10
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, antibodies are expensive, poorly stable, and highly immunogenic, and the method is time-consuming, complicated to operate, and prone to false positive results
Detection methods using nucleic acid aptamers as recognition units can achieve highly specific and sensitive detection of ricin, but some methods have the disadvantages of time-consuming and cumbersome operations
In addition, these two types of methods will use expensive and cumbersome laboratory instruments such as mass spectrometry (MS), atomic force microscopy (AFM), and surface-enhanced Raman spectroscopy (SERS), which are difficult to achieve instant detection in the field, at home, and in resource-poor areas ( POCT)

Method used

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  • Method for detecting chain B of ricin in real time by using dual amplification strategy of catalytic hairpin assembly mediated lipidosome encoding magnetic beads
  • Method for detecting chain B of ricin in real time by using dual amplification strategy of catalytic hairpin assembly mediated lipidosome encoding magnetic beads
  • Method for detecting chain B of ricin in real time by using dual amplification strategy of catalytic hairpin assembly mediated lipidosome encoding magnetic beads

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation and Characterization of Glucose Oxidase Liposomes

[0047] First, soybean lecithin, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000 ) respectively according to 18mg, 4.6mg, 3.3mg (molar ratio 100:50:5) into the round bottom flask and dissolved with 1mL chloroform. Then, chloroform was removed under reduced pressure in a rotary evaporator at 40° C. for 30 minutes to obtain a film, and then vacuum-dried for 2 hours to remove residual organic solvent. Then, 2 mL of PBS (0.01 M, pH 7.4) solution containing 5 mg / mL GOD was added to the dried film, hydrated at 40 °C for 30 minutes to form a cloudy suspension, and then passed through a 100 nm polycarbonate membrane to obtain liposomes. Finally, a 300 kDa dialysis bag was used to stir in PBS (0.01 M, pH 7.4) at 4° C. for 24 h to remove uncoated glucose oxidase. Meanwhile, the obtained liposomes were stored at 4°C for subsequent use. T...

Embodiment 2

[0048] Embodiment 2: Preparation of magnetic beads-nucleic acid aptamer-blocker hybridization probe and MBs-H1 and H2-L GOD

[0049] (1) Preparation of magnetic bead-adapter-blocker hybridization probe: First, add the nucleic acid aptamer of ricin B chain, blocker and 10×TNaK solution together, and anneal the mixture at 95°C for 5 minutes Let stand for 30 minutes to cool to room temperature. Subsequently, streptavidin-modified magnetic beads (resuspended in 1×TNaK solution) were added to the above mixture and incubated at room temperature for 30 min. After removing the supernatant, the magnetic beads (MBs-aptamer-blocker ) three times and resuspended in 1×TNaK buffer.

[0050] (2) Preparation of MBs-H1: First, heat the biotin-modified H1 at the 3' end to 95°C for 1 minute and then let it stand for 30 minutes to cool to room temperature, then add streptavidin-modified magnetic beads (resuspended in 1×TNaK solution) and incubate at room temperature for 30 minutes with shaking...

Embodiment 3

[0052] Example 3: CHA-mediated liposome-encoded magnetic bead strategy for the detection of RTB

[0053] Add RTB and TNaK buffer to the magnetic bead-aptamer-blocker hybridization probe, incubate at room temperature for 40 minutes, and magnetically separate the supernatant, which is the blocker, and participate in the next step of the experiment. Then, MBs-H1, H2-L GOD MBs-H1-H2-L obtained after reacting with blocker at 37°C for 1 hour, washing with PBS three times to remove unreacted components GOD . Subsequently, 10 mM glucose solution (1% Triton X-100, 10 mM PBS, pH 5.5) was added to MBs-H1-H2-L GOD In , after reacting at 55 °C for 40 minutes, the liposome membrane was destroyed to release GOD, which catalyzed the oxidation of glucose to produce H 2 o 2 . Finally, through PTS and handheld H 2 o 2 The detector can realize naked-eye qualitative and quantitative detection of B chain at the same time.

[0054] See Table 1 for the nucleotide sequences involved in the pre...

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Abstract

The invention relates to a method for detecting chain B of ricin in real time by using a dual amplification strategy of catalytic hairpin assembly mediated lipidosome encoding magnetic beads. The method comprises the following steps: firstly, releasing a blocker through incubation of a magnetic bead-nucleic acid aptamer-blocker hybrid probe and chain B of ricin, secondly, enabling H1 modified magnetic beads, H2 functionalized lipidosome and the blocker so as to obtain MBs-H1-H2-LGOD, performing magnetic separation, adding a glucose solution into MBs-H1-H2-LGOD, breaking a lipidosome membrane through the reaction to release coated GOD (glucose oxidase) for catalytic oxidation on glucose to generate H2O2, developing the blue color (qualitative) of H2O2 by using PTS (peroxide test), and reading values (quantitative) by using a handheld H2O2 detector, so as to achieve simultaneous naked-eye qualitative and quantitive detection on the chain B of the ricin. The method provided by the invention has super high sensitivity and selectivity, and is simple and convenient and low in cost.

Description

technical field [0001] The invention belongs to the development of new analysis and sensing technology, and relates to a method for instant detection of ricin B chain by catalyzing hairpin assembly and mediating a double amplification strategy of liposome-encoded magnetic beads. Background technique [0002] Ricin, a type II ribosome-inactivating protein (RIP II) extracted from castor beans, is one of the deadliest known toxins. Ricin is composed of A and B chains linked by disulfide bonds. After the B chain (RTB) combines with glycoproteins or glycolipids on the cell membrane, the A chain is separated from the B chain and enters the ribosome in the cell to play a toxic role. , which can inactivate 1500 ribosomes in 1 minute thereby inhibiting protein synthesis and eventually leading to cell death. The median lethal dose (LD) of ricin to the human body through inhalation 50 ) is about 5-10 μg / kg, by ingesting about 1-20 mg / kg or 8 castor seeds, and there is currently no ef...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682C12Q1/26G01N21/78
CPCC12Q1/682C12Q1/26G01N21/78C12Q2525/205C12Q2525/301C12Q2563/143C12Q2563/149
Inventor 甄淑君门晨
Owner SOUTHWEST UNIVERSITY
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