Quantitative detection method for quorum sensing signal molecules AHLs in sludge phase

Abstract

The invention provides a quantitative detection method for quorum sensing signal molecules AHLs in a sludge phase. The quantitative detection method comprises the following steps: step S1, sample pretreatment; s2, performing LC-MS (liquid chromatography-mass spectrometry) or MS detection optimization; and S3, whole-process verification is carried out. According to the quantitative detection method for quorum sensing signal molecules AHLs in the sludge phase, the technical bottleneck of a traditional method in sample pretreatment and matrix effect is broken through, and a reliable tool is provided for QS mechanism research and sewage system optimization.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of environmental microbial detection and growth, and particularly relates to a quantitative detection method for quorum sensing signal molecules AHLs in sludge phase. BACKGROUND

[0002] Quorum sensing (QS) is a core mechanism of microbial regulation of group behavior (biofilm formation, pollutant degradation, etc.) through the secretion of signal molecules (such as AHLs). In biological wastewater treatment systems, QS directly affects membrane fouling in membrane bioreactors (MBRs), stability of aerobic granular sludge (AGS), and nitrogen conversion efficiency.

[0003] Quorum sensing (QS) has the following technical defects:

[0004] (1) AHLs have strong hydrophobicity (increasing with the number of side chain carbon atoms), which causes them to mainly accumulate in sludge flocs rather than in the aqueous phase in wastewater treatment systems;

[0005] (2) Traditional detection methods (liquid-liquid extraction or solid-phase extraction + LC-MS or MS) have two major defects:

[0006] (3) The extraction recovery rate of AHLs in sludge phase is low;

[0007] (4) The matrix effect is significant in LC-MS or MS detection, leading to quantitative bias;

[0008] (5) There is no standardized method to completely detect 11 trace AHLs (detection limit needs to reach ng or L level) in sludge phase. SUMMARY

[0009] In view of the above, in order to overcome the defects of the prior art, the present application provides a quantitative detection method for quorum sensing signal molecules AHLs in sludge phase, which breaks through the technical bottleneck of traditional methods in sample pretreatment and matrix effect, and provides a reliable tool for QS mechanism research and wastewater system optimization.

[0010] To achieve the above purpose, the present application provides the following technical scheme: a quantitative detection method for quorum sensing signal molecules AHLs in sludge phase, comprising,

[0011] Step S1, sample pretreatment;

[0012] Step S2, LC-MS or MS detection optimization;

[0013] Step S3, full-process verification. The present application provides a quantitative detection method for quorum sensing signal molecules AHLs in sludge phase, which breaks through the technical bottleneck of traditional methods in sample pretreatment and matrix effect, and provides a reliable tool for QS mechanism research and wastewater system optimization.

[0014] Optionally, step S1 may further include:

[0015] Step S1.1, Sludge floc structure disruption process: Ultrasonic-chemical synergistic disruption method is used to release AHLs embedded in the flocs;

[0016] Step S1.2, Targeted enrichment extraction: Optimize the combination of solid phase extraction adsorbents to improve the recovery rate of hydrophobic AHLs;

[0017] Step S1.3, Internal Standard Correction: Add isotope-labeled AHLs internal standards (e.g., ), to compensate for matrix effects in real time.

[0018] Optionally, an ultrasonic-chemical synergistic disruption method is used, employing EDTA combined with enzymatic hydrolysis for release.

[0019] Optionally, the internal standard for isotope-labeled AHLs is as follows: .

[0020] Optionally, step S2 may further include the following steps:

[0021] Step S2.1, Chromatographic separation: A reverse gradient elution program is used to accurately separate isomers;

[0022] Step S2.2, Mass spectrometry detection: Establish a multi-reaction monitoring ion pair database covering the characteristic parent ion or daughter ion pairs of 11 AHLs;

[0023] Step S2.3, Quantitative model: Construct a calibration curve based on the internal standard method, with a dynamic range of 0.1–1000 ng or L and a detection limit of ≤0.05 ng or L.

[0024] Optionally, the isomer is... and .

[0025] Optionally, step S3 may also include:

[0026] Step S3.1, spike recovery rate: 85%~115%;

[0027] Step S3.2, relative standard deviation: <5%.

[0028] In summary, the beneficial effects of this invention are:

[0029] This invention provides a quantitative detection method for quorum sensing signal molecules (AHLs) in sludge phases, overcoming the technical bottlenecks of traditional methods in sample pretreatment and matrix effects, and providing a reliable tool for QS mechanism research and wastewater system optimization. Detailed Implementation

[0030] In the description of this specification, the references to terms such as "certain embodiments," "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0031] In this invention, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance; the term "multiple" refers to two or more unless otherwise explicitly defined. The terms "install," "connect," "link," and "fix" should be interpreted broadly. For example, "connect" can be a fixed connection, a detachable connection, or an integral connection; "link" can be a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0032] Example

[0033] This embodiment provides a method for the quantitative detection of quorum sensing signal molecules (AHLs) in sludge phases, comprising the following steps:

[0034] Step S1, sample pretreatment;

[0035] Step S2, LC-MS or MS detection optimization;

[0036] Step S3, full-process verification.

[0037] Step S1 further includes the following steps:

[0038] Step S1.1, Sludge floc structure disruption process: Ultrasonic-chemical synergistic disruption method is used to release AHLs embedded in the flocs;

[0039] Step S1.2, directional enrichment extraction: optimize the solid phase extraction (SPE) adsorbent combination to improve the recovery rate of hydrophobic AHLs;

[0040] Step S1.3, Internal Standard Correction: Add isotope-labeled AHLs internal standards (e.g., ), to compensate for matrix effects in real time.

[0041] Furthermore, step S2 also includes the following steps:

[0042] Step S2.1, Chromatographic separation: A reverse gradient elution program is used to accurately separate isomers (such as...). and )

[0043] Step S2.2, Mass spectrometry detection: Establish a multiple reaction monitoring (MRM) ion pair database covering the characteristic parent ion or daughter ion pairs of 11 AHLs;

[0044] Step S2.3, Quantitative model: Construct a calibration curve based on the internal standard method, with a dynamic range of 0.1–1000 ng or L and a detection limit of ≤0.05 ng or L.

[0045] Furthermore, step S3 also includes the following steps:

[0046] Step S3.1, spike recovery rate: 85%~115%;

[0047] Step S3.2, Relative Standard Deviation (RSD): <5% (within or between batches).

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that modifications or equivalent substitutions to the technical solutions of the present invention do not depart from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for quantitative detection of quorum sensing signal molecules (AHLs) in sludge phase, characterized in that, include, Step S1, sample pretreatment; Step S2, LC-MS or MS detection optimization; Step S3, full-process verification.

2. The method for quantitative detection of quorum sensing signal molecules AHLs in sludge phase according to claim 1, characterized in that, Step S1 also includes: Step S1.1, sludge floc structure disruption process, using ultrasonic-chemical synergistic disruption method to release AHLs embedded in the flocs; Step S1.2: Targeted enrichment extraction, optimizing the combination of solid-phase extraction adsorbents to improve the recovery rate of hydrophobic AHLs; Step S1.3, internal standard correction, add isotope-labeled AHLs internal standards (such as ¹³C6-AHLs) to compensate for matrix effects in real time.

3. The method for quantitative detection of quorum sensing signal molecules AHLs in sludge phase according to claim 2, characterized in that, The ultrasonic-chemical synergistic disruption method uses EDTA combined with enzymatic hydrolysis for release.

4. The method for quantitative detection of quorum sensing signal molecules AHLs in sludge phase according to claim 2, characterized in that, The internal index for isotopic AHLs is ¹³C6-AHLs.

5. The method for quantitative detection of quorum sensing signal molecules AHLs in sludge phase according to claim 1, characterized in that, Step S2 also includes the following steps: Step S2.1, Chromatographic separation: A reverse gradient elution program is used to accurately separate isomers; Step S2.2, Mass spectrometry detection: Establish a multi-reaction monitoring ion pair database covering the characteristic parent ion or daughter ion pairs of 11 AHLs; Step S2.3, Quantitative model: Construct a calibration curve based on the internal standard method, with a dynamic range of 0.1–1000 ng or L and a detection limit of ≤0.05 ng or L.

6. The method for quantitative detection of quorum sensing signal molecules AHLs in sludge phase according to claim 5, characterized in that, The isomers are C4-HSL and 3OC4-HSL.

7. The method for quantitative detection of quorum sensing signal molecules AHLs in sludge phase according to claim 1, characterized in that, Step S3 also includes: Step S3.1, spike recovery rate: 85%~115%; Step S3.2, relative standard deviation: <5%.