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Method for simultaneously determining bacterial quorum sensing AHLs molecules by using high performance liquid chromatography-tandem mass spectrometry method

A technology of high-performance liquid chromatography and tandem mass spectrometry, which is applied in the field of determination of bacterial quorum-sensing secretion of AHLs-like signal molecules, can solve the problems of cumbersome operation, low sensitivity, and long analysis time of detection methods, and achieve convenient analysis methods, high sensitivity, The effect of easy operation

Inactive Publication Date: 2013-04-24
SHANGHAI OCEAN UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

Among them, the TLC-Biosensor method is widely used at present, but different bacterial biosensors have different sensitivity to AHLs with different acyl side chain lengths, for example, Agrobacterium tumefaciens is sensitive to AHLs with C-3 carbonyl substitution of acyl side chains The strongest, and purple bacteria (chromobacterium violaceum CVO26) is more sensitive to short-chain C-3 AHLs without substituents, so the type of AHLs cannot be accurately characterized, and this method cannot be used for precise quantitative analysis
The rapid development of GC-MS and HPLC-MS analysis technology can qualitatively analyze the AHLs released by bacteria, but it cannot meet the needs of experiments in mixed sample injection and microquantitative analysis
At present, there are few reports on the qualitative and quantitative analysis of AHLs signal molecules by HPLC-MS / MS. Daniele Morin et al. used HPLC-MS / MS to analyze AHLs signal molecules, but the detection method takes a long time to analyze. (about 40min), cumbersome operation and low sensitivity

Method used

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  • Method for simultaneously determining bacterial quorum sensing AHLs molecules by using high performance liquid chromatography-tandem mass spectrometry method
  • Method for simultaneously determining bacterial quorum sensing AHLs molecules by using high performance liquid chromatography-tandem mass spectrometry method
  • Method for simultaneously determining bacterial quorum sensing AHLs molecules by using high performance liquid chromatography-tandem mass spectrometry method

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Experimental program
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Effect test

Embodiment 1

[0021] The pretreatment of embodiment 1 sample

[0022] A single colony of Pseudomonas fluorescens was cultured in a 250mL Erlenmeyer flask containing 50mL LB medium, and cultured on a shaker at 28°C for 12h to prepare a seed culture solution. After culturing for 12 hours, take 1 mL of the seed culture solution and culture it in a 500 mL Erlenmeyer flask containing 100 mL of LB culture solution for 6, 10 and 12 hours respectively.

[0023] Put the above culture solution in a 50mL centrifuge tube and centrifuge at 27000g for 20min at 4°C. The supernatant was extracted three times with an equal amount of ethyl acetate containing 0.02mol / L glacial acetic acid, the aqueous phase was discarded, and the organic phase was mixed. The ethyl acetate extract obtained above was placed in a rotary evaporator and evaporated to dryness. Dissolve the evaporated flask above with anhydrous methanol, and dilute to 5 mL. Take 0.5mL of this solution, add 0.5mL of ultrapure water, pass through a...

Embodiment 2

[0024] Example 2 Simultaneous Determination of Bacterial Quorum Sensing AHLs Signaling Molecules by High Performance Liquid Chromatography-Tandem Mass Spectrometry

[0025] 1. Chromatography and mass spectrometry conditions:

[0026] Column: Phenomenex C 18 (2.0mm×100mm3μm); flow rate: 0.2mL / min; injection volume: 10μL; analysis time: 15min; Concentration) methanol of formic acid; Phase B is an aqueous phase, which is an aqueous solution containing 10mmol / L ammonium acetate and 0.2% (volume concentration) formic acid. See Table 1 for gradient conditions.

[0027] Liquid phase gradient: This method uses Waters2695Quattro micro liquid chromatography tandem mass spectrometry instrument with 11 gradient curve types, such as Figure 9 shown. The curve can be changed in the inlet method editor to realize the adjustment of the liquid phase gradient.

[0028] Curve 1 indicates that the set ratio of the two mobile phases for cleaning the chromatographic column is reached instantly...

Embodiment 3

[0042] Prepare eleven kinds of AHLs signal molecules 1, 10, 100, 250ng / L mixed standard solutions (concentrations of each signal molecule are 1, 10, 100, 250ng / L respectively) for determination to quantify ions (m / z102) The peak area (Y) and the corresponding standard concentration (X, ng / L) were used as a standard working curve. The correlation coefficients r of the 11 AHLs signal molecules were all greater than 0.996, and there were good results in the range of 1 (10) to 250ng / L. linear relationship. The detection limit and quantification limit are obtained by the calculation method of S / N=3, S / N=10, and the results are shown in Table 4. The chromatograms of 11 kinds of AHLs signal molecule standards are as follows Figure 1 ~ Figure 3 shown. Four AHLs (3-oxo-C 6 -HSL, 3-oxo-C 10 -HSL, C 4 -HSL and C 8 -HSL) Secondary mass spectrogram such as Figure 4 ~ Figure 7 shown. The detection result is the same as the standard spectrum.

[0043] Table 411 kinds of AHLs signal ...

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Abstract

The invention relates to the field of analytical chemistry, and discloses a method for simultaneously determining bacterial quorum sensing AHLs molecules. A to-be-detected sample is added in a high performance liquid chromatography-tandem mass spectrometer for gradient elution. The elution conditions are that the elution is carried out by using an A phase (methanol containing 10 mmoI / L ammonium acetate and 0.2 % formic acid) and a B phase (a water solution containing 10 mmoI / L ammonium acetate and 0.2 % formic acid); parameters for the gradient elution are as follows: at 0 min, the content of the A phase is 20% of and the content of the B phase is 80% and the content reaches a preset proportion at the moment; from 0 min to 8 min, two flowing phases are changed into 100% of the A phase and 0% of the B-phase at an average rate; from 8 min to 10.1 min, the content of the A phase is 100% of and the content of the B phase is 0%; at 10.1 min, the content of the A phase is adjusted to 20% of and the content of the B phase adjusted to 80% instantly; and elution is continuously performed to 15 min. The method has good linear relationship, a detection unit of 0.1-1.0 ng / L, has high accuracy and sensitivity, and simple operations.

Description

technical field [0001] The invention relates to the field of analytical chemistry, in particular to a measurement method for bacterial quorum sensing and secretion of AHLs signal molecules. Background technique [0002] Signaling molecules (N-acyl-homoserine lactones, N-acyl-homoserine lactones, referred to as AHLs), also known as autoinducers, are the key substances for the quorum sensing effect (Quorum sensing) between microorganisms. Microorganisms secrete signal molecules, Release and induction carry out intra-species and inter-species communication and complete the expression of certain characteristics. The quorum sensing effect means that microbial cells sense the concentration of extracellular small molecule autoinducers through the corresponding sensing system, thereby sensing the size of the bacterial population density. When the bacterial population density reaches a certain threshold, a series of target genes are activated and the corresponding characteristics ar...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/34
Inventor 马晨晨欧杰李柏林王婧
Owner SHANGHAI OCEAN UNIV
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