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Method for detecting endotoxin

An endotoxin and sample technology, which can be used in measurement devices, biological tests, material inspection products, etc., can solve the selectivity and sensitivity limitations of graphene sensors, cannot control reaction conditions well, and graphene has low biocompatibility, etc. problem, to achieve the effect of strong anti-interference ability, short time and less consumption of reagents

Active Publication Date: 2019-01-25
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For graphene, if the sensor is only made of graphene, the reaction conditions cannot be well controlled
For example, graphene has very low biocompatibility, which affects its biorecognition process
On top of that, the selectivity and sensitivity of graphene sensors are limited

Method used

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Examples

Experimental program
Comparison scheme
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Embodiment 1

[0036] Embodiment 1 A kind of method for detecting endotoxin

[0037] 1) Fabrication of a microfluidic chip: In this embodiment, a PDMS (polydimethylsiloxane) microfluidic chip is used, which contains a Nafion membrane, and the Nafion membrane is perpendicular to the channel in the microfluidic chip. The specific fabrication process of the microfluidic chip is as follows:

[0038]Treat the silicon wafer with trimethylchlorosilane in a vacuum desiccator to avoid adhesion of PDMS to the silicon wafer mold. PDMS and curing agent are fully mixed at a weight ratio of 10:1, placed in a vacuum desiccator for vacuum degassing until the bubbles completely disappear, and then slowly poured on the silicon wafer to avoid bubbles. Then cure on a heating plate at 95°C for 2-3h, and then peel off the PDMS from the silicon wafer, so that the microstructure on the silicon wafer is transferred to the elastic PDMS, and holes are punched at the inlet and outlet . Then make Nafion membrane on t...

Embodiment 2

[0044] Example 2 A method for detecting endotoxin (optimization of heating time, ice bath time and incubation time with rGO of LPS aptamer)

[0045] 1) Fabrication of the microfluidic chip: same as in Example 1

[0046] 2) Sample processing:

[0047] Preparation of LPS standard solution: Prepare a standard stock solution (1 mg / mL) of LPS with ultrapure water. LPS solutions with different concentrations were prepared by diluting stock solutions appropriately. In this embodiment, 10 pM LPS solution is used as the subsequent sample to be tested.

[0048] Take the 6-FAM (6-carboxyfluorescein)-labeled LPS aptamer (designed and synthesized by the biological company) and heat it in a 95°C water bath for 1, 5, 10, 12, and 20 minutes, and then immediately put it into 15°C water and ice-bath it separately. 1, 5, 10, 12, 20 minutes. Then it was mixed with the sample to be tested (10 pM LPS solution, the final concentration of LPS was 1 pM) and the first incubation was continued at 15...

Embodiment 3

[0053] Embodiment 3 A kind of method for detecting endotoxin (CH 3 CN effect test)

[0054] 1) Fabrication of the microfluidic chip: same as in Example 1

[0055] 2) Sample processing:

[0056] Preparation of LPS spiked serum: Collect serum from healthy people, add LPS standard stock solution of known concentration in Example 2, heat the mixture in boiling water at 95°C for 5 minutes to obtain LPS spiked serum, add the spiked serum to SDS 10mg / mL, β-mercaptoethanol 5% v / v, and heated in boiling water at 95°C for 5min, as the subsequent sample to be tested.

[0057] Take the 6-FAM (6-carboxyfluorescein)-labeled LPS aptamer (designed and synthesized by Biological Company) and heat it in a water bath at 95°C for 10 minutes, and then put it into water at 15°C for 10 minutes on ice respectively. Then mix it with the above-mentioned sample to be tested and continue to incubate for the first time at 15° C. for 10 min. Then, the reduced graphene oxide rGO and 1×TBE buffer were add...

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Abstract

The invention discloses a method for detecting endotoxin. The method comprises the following steps of performing incubation on a to-be-detected sample and a fluorescently-labeled LPS aptamer; then adding reduced graphene oxide and a TBE buffer solution, and performing incubation; adding the incubated sample into a sample inlet of a micro-fluidic chip, applying a voltage between the sample inlet and a sample outlet of the micro-fluidic chip to perform processing, arranging an anode at the sample inlet, and detecting the fluorescence intensity between a Nafion membrane channel and the anode after the voltage processing; and determining the content of LPS in the to-be-detected sample according to the fluorescence intensity and a standard curve. The method has a lowest detection limit of 8 fM,is wide in linear range, high in sensitivity, good in selectivity and relatively strong in anti-interference capability, and can rapidly distinguish gram-positive bacteria, gram-negative bacteria andfungi.

Description

technical field [0001] The invention relates to a method for detecting endotoxin. Background technique [0002] Endotoxin (LPS) is a highly toxic inflammatory stimulus that can interact with specific cell receptors to produce inflammatory cytokines that can lead to fever, sepsis, multiple organ failure and even death. Due to the possibility of severe immune reactions, the detection of LPS has become critical, especially for biological products such as injections, in order to ensure the safety of sterilized products. At present, the main method used for LPS detection is the limulus assay (LAL), which is widely used in many fields. [2] In recent years, there have been some studies on electrochemical and optical sensors based on LPS antibodies or aptamers, [3,4] They can specifically identify LPS and overcome some of the shortcomings of traditional methods. Aptamers are single-stranded oligonucleotides (ssRNA or ssDNA) with high specificity and high affinity for target bindi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/53
CPCG01N33/5302G01N33/533
Inventor 刘利红牛俊心
Owner SOUTHERN MEDICAL UNIVERSITY
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