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Fluorescent staining reagent for detecting the dead and alive state of bacteria and fungi, preparation method and application

A fluorescent dyeing and reagent technology, applied in the field of microbial detection, can solve the problems of large biological toxicity, poor water solubility, deviation of test results, etc., and achieve the effect of good water solubility, safe composition and low toxicity

Active Publication Date: 2018-07-20
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the molecule has great biological toxicity and has potential harm to the human body and the ecological environment.
[0005] In addition, the detection method based on propidium iodide requires observation within a short time after the bacteria are stained, otherwise the detection results will be biased
Due to the large number of benzene ring structures, the water solubility of such dye molecules is very poor, and organic solvents such as dimethyl sulfoxide must be used to dissolve them

Method used

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  • Fluorescent staining reagent for detecting the dead and alive state of bacteria and fungi, preparation method and application
  • Fluorescent staining reagent for detecting the dead and alive state of bacteria and fungi, preparation method and application
  • Fluorescent staining reagent for detecting the dead and alive state of bacteria and fungi, preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0027] (1) First, protoporphyrin (PpIX), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS ) is activated as a raw material to the carboxyl group of the protoporphyrin molecule. Weigh 10 mg PpIX, 20.4 mg EDC·HCl and 12.3 mg NHS and dissolve them in 1 mL dimethyl sulfoxide (DMSO). PpIX is first mixed with EDC·HCl and then NHS is added to react at room temperature for 2 hours to fully activate the carboxyl group.

[0028] (2) Weigh 4.2 mg of cholesterol-PEG2000-amino (cholesterol-PEG2000-NH 2 ) and 43 μL triethylamine were co-dissolved in DMSO and mixed with the above reaction system. After overnight reaction at room temperature, purification was performed by dialysis against DMSO for 2 days using a dialysis bag with a molecular weight cut-off of 2k, followed by dialysis in ultrapure water for 1 day. Finally, it was freeze-dried in a lyophilizer to produce cholesterol-PEG2000-PpIX, and stored at -20°C.

[0029] The reagent obt...

Embodiment 201

[0031] In Example 201, the molecular weight of polyethylene glycol is 1000, and the fluorescent molecules protoporphyrin (PpIX), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl ) and N-hydroxysuccinimide (NHS) in a molar ratio of 1:4:48; the reaction time at room temperature is 3 hours; the molar ratio of fluorescent molecules to cholesterol-polyethylene glycol-amino molecules is 2:1. The MWCO of the dialysis bag is 1000.

Embodiment 202

[0032] In Example 202, the molecular weight of polyethylene glycol is 5000, and the fluorescent molecules protoporphyrin (PpIX), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl ) and N-hydroxysuccinimide (NHS) in a molar ratio of 1:10:20; the reaction time at room temperature is 2 hours; the molar ratio of fluorescent molecules to cholesterol-polyethylene glycol-amino molecules is 10:1. The MWCO of the dialysis bag is 1000.

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Abstract

The invention discloses a fluorescent dyeing reagent for detecting a bacterium and fungus survival state and its preparation method and use. The fluorescent dyeing reagent comprises dye molecules. The dye molecule is prepared through respectively connecting a cholesterol molecule and a fluorescent molecule to two ends of a polyethylene glycol molecule. The fluorescent dyeing reagent has low bacterium or animal cell toxicity, has little biotoxicity and realizes long-term detection of a bacterium and fungus survival state. The fluorescent dyeing reagent has good water solubility.

Description

technical field [0001] The invention belongs to the field of microorganism detection, in particular to a fluorescent dyeing reagent for detecting the dead and alive states of bacteria and fungi and a method for preparing the fluorescent dyeing reagent. Background technique [0002] Microbiological detection plays an important role in environmental monitoring, medical and health, food processing, and pharmaceutical industries. Among them, the life and death identification of microorganisms is a key detection index in reflecting the quality of drinking water and food, evaluating the effect of antibacterial materials and determining the source of pollution. The traditional plate count method evaluates the number of live bacteria in a sample by utilizing the ability of live bacteria to reproduce on a culture plate. [0003] In this method, one or a small number of viable bacteria will grow as a macroscopic plaque on a suitable agarose culture plate, so the number of colony form...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C09K11/06C09B47/00G01N21/64
CPCC09B47/00C09K11/06C09K2211/1029G01N21/6486
Inventor 吴富根贾浩然祝雅璇
Owner SOUTHEAST UNIV
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