阅读说明：本技术 一种基于等离激元金纳米囊泡的超灵敏细菌鉴定方法 (Ultra-sensitive bacterium identification method based on plasmon gold nano vesicles ) 是由 乔亮 易佳 王新军 刘宝红 于 2019-10-15 设计创作，主要内容包括：本发明公开了一种基于等离激元金纳米囊泡的超灵敏细菌鉴定方法。该方法利用等离激元金纳米囊泡包封细菌样品,将细菌样品限制在直径为100微米的等离激元金纳米囊泡中,通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术进行细菌鉴定。本发明构建的细菌鉴定方法,能够实现低至40个细菌的细菌鉴定,具有较强的可操作性和实用性。(The invention discloses an ultrasensitive bacteria identification method based on plasmon gold nano vesicles. The method comprises the steps of encapsulating a bacterial sample by utilizing plasmonic gold nanovesicles, limiting the bacterial sample in the plasmonic gold nanovesicles with the diameter of 100 microns, and carrying out bacterial identification by a matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) technology. The bacteria identification method constructed by the invention can realize the bacteria identification of as few as 40 bacteria, and has stronger operability and practicability.)

1. An ultrasensitive bacteria identification method based on plasmon gold nano vesicles is characterized by comprising the following specific steps:

(1) uniformly mixing the bacteria sample and the matrix solution;

(2) synthesizing gold nanoparticle sol modified by perfluorodecyl mercaptan;

(3) adding the bacterial sample uniformly mixed with the matrix solution obtained in the step (1) into the sol synthesized in the step (2), oscillating and emulsifying to obtain plasmon gold nano vesicles encapsulating the bacterial sample, and moving the single vesicles out of the target plate to obtain sample points close to the laser spot size;

(4) analyzing the sample points obtained in the step (3) by adopting matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) to obtain a bacterial mass spectrogram;

(5) and (4) comparing the bacterial mass spectrum obtained in the step (4) with a standard bacterial mass spectrum database to obtain an identification result.

2. The method of claim 1, wherein: the diameter of the gold nano particles modified by the perfluorodecyl mercaptan in the step (2) is 25-35 nanometers, and the concentration is 35 picomoles per liter.

3. The method according to claim 1, wherein the size of the plasmonic gold nanovesicle in the step (3) is 20 ~ 500 microns.

4. The method of claim 1, wherein: the matrix solution in the step (1) is prepared from a mixed solvent consisting of alpha-cyano-4-hydroxycinnamic acid, acetonitrile, trifluoroacetic acid and deionized water, wherein: the volume ratio of the acetonitrile to the trifluoroacetic acid to the deionized water is 50:2.5: 47.5; the alpha-cyano-4-hydroxycinnamic acid is a saturated solution.

5. The method of claim 1, wherein: and (2) uniformly mixing the bacteria sample and the matrix solution in the step (1) by adopting a vortex mixer.

6. The method of claim 1, wherein: the standard bacteria mass spectrum database in the step (5) is any one of a Biotyper microbial database or a VITEK MS database.

Technical Field

The invention belongs to the field of medical detection, and particularly relates to an ultrasensitive bacterium identification method based on plasmon gold nano vesicles.

Background

Bacterial resistance causes high morbidity, high mortality, and carries a high economic burden. The rapid identification of bacteria plays an important role in the diagnosis of bacterial infections. Bacteria identification methods are many, such as biochemical assays, immunological methods, DNA sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Wherein the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF)MS) has completely changed the current situation of bacterial identification, and a bridge is established between the genotype and the phenotype of bacteria. [ BIZZINI, A., GRUB, G. Matrix Assisted Laser Desorption ionization Time of Flight Mass Spectrometry a reselection in clinical Microbiological identification ] [ J]. Clinical Microbiology and Infection, 2010, 16,1614-1619.]However, when the bacteria identification is carried out by using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a single sample point needs to exceed 10⁴The detection sensitivity of matrix-assisted laser desorption ionization time-of-flight mass spectrometry is greatly limited by a sample preparation method, the traditional sample preparation method is to directly perform sample application on a common target plate by using a liquid-moving gun, and the diameter of an obtained sample point is about 2 mm and is far larger than the diameter of a laser beam (~ 0.1.1 mm), so that the sample cannot be effectively utilized.

The plasmon nanometer vesicle is a microcapsule, namely a layer of self-assembled nanometer particles is coated on the surface of a micron-sized liquid drop. The method can rapidly prepare a large amount of micron-sized plasmon nanovesicles with certain sizes only by simple emulsification operation, wherein the method comprises the steps of [ DINSMORE, A, D, HSU.M. F., NIKOLAIDES, M.G., et al, colloids: selective Permeable Capsules compounded of Colloidal particles [ J ]. Science,2002, 298: 1006-. The diameter of [ BOLLHORST, T., REZWAN, K., MAAS, M., et al, Colloidal capsules: Nano-and Microcapsules with Colloidal Particle shells [ J ]. Chemical Society Reviews, 2017, 46, 2091-2126 ] plasmonic nanovesicles can be matched to the diameter of the laser beam of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Therefore, by utilizing the characteristic, the plasmon nano vesicles are applied to the sample preparation process of MALDI-TOF MS, small-size sample points are generated at high throughput, and the sample points enable a bacteria sample to have very high surface density, so that the sample utilization rate in the ionization/desorption process is greatly improved, and the sensitivity of MALDI-TOF MS for detecting bacteria is improved.

Disclosure of Invention

In order to further improve the sensitivity of identifying bacteria by MALDI-TOF MS and overcome the defects of the traditional sample preparation method, the invention aims to provide an ultrasensitive bacteria identification method based on plasmon gold nano vesicles. The method can realize the rapid identification of the bacteria with low copy number, has simple and efficient preparation process, and greatly improves the detection sensitivity compared with the traditional MALDI-TOF MS method. Through the bacterium of plasmon gold nanometer vesicle encapsulation, only the sample of nanometer upgrading is used for the analysis, and remaining can be retrieved through centrifugal action and be used for other experiments, effectively saves the sample.

The technical scheme of the invention is as follows:

an ultrasensitive bacteria identification method based on plasmon gold nano vesicles comprises the following specific steps:

(1) uniformly mixing the bacteria sample and the matrix solution;

(2) synthesizing gold nanoparticle sol modified by perfluorodecyl mercaptan;

(3) adding the bacterial sample uniformly mixed with the matrix solution obtained in the step (1) into the sol synthesized in the step (2), oscillating and emulsifying to obtain plasmon gold nano vesicles encapsulating the bacterial sample, and moving the single vesicles out of the target plate to obtain sample points close to the laser spot size;

(4) analyzing the sample points obtained in the step (3) by adopting matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) to obtain a bacterial mass spectrogram;

(5) and (4) comparing the bacterial mass spectrum obtained in the step (4) with a standard bacterial mass spectrum database to obtain an identification result.

In the invention, the gold nanoparticles modified by the perfluorodecyl mercaptan (perfluorodecyl mercaptan-gold nanoparticles) in the step (2) have a diameter of 25-35 nm and a concentration of 35 picomoles per liter.

In the present invention, the size of the plasmonic gold nanovesicle in step (3) is 20 ~ 500 micrometers.

In the invention, the matrix solution in the step (1) is prepared from a mixed solvent consisting of alpha-cyano-4-hydroxycinnamic acid, acetonitrile, trifluoroacetic acid and deionized water, wherein: the volume ratio of the acetonitrile to the trifluoroacetic acid to the deionized water is 50:2.5: 47.5; the alpha-cyano-4-hydroxycinnamic acid is a saturated solution.

In the invention, the bacteria sample and the matrix solution in the step (1) are uniformly mixed by adopting a vortex mixer.

In the present invention, the standard bacterial mass spectrum database in step (5) is any one of a Biotyper microbial database and a VITEKMS database.

In the invention, when matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology is adopted for detection, a matrix solution is prepared by a mixed solvent consisting of alpha-cyano-4-hydroxycinnamic acid and acetonitrile, trifluoroacetic acid and deionized water, wherein the volume ratio of the acetonitrile to the trifluoroacetic acid to the deionized water is 50:2.5: 47.5; the alpha-cyano-4-hydroxycinnamic acid is a saturated solution. Before the bacterial sample is encapsulated in the plasmon gold nano vesicles, a vortex mixer is used for uniformly mixing the bacterial suspension and the matrix solution.

Compared with the prior art, the invention has the beneficial effects that: the method has strong operability and practicability, the low-copy-number bacteria sample is packaged by utilizing the plasmon gold nano vesicles, and the bacteria packaged in the low-copy-number bacteria sample is identified by adopting a matrix assisted laser desorption ionization time-of-flight mass spectrometry technology. The sensitivity of the method can be tested to be as low as 40 bacteria.

Drawings

FIG. 1: schematic diagram of experimental basic flow.

FIG. 2: and mass spectrograms of 4 different pathogenic bacteria encapsulated in the plasmon gold nano vesicles.

FIG. 3 Standard spectrum of 4 pathogenic bacteria (~ 10)⁶Individual bacteria/sample well).

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention is further described below with reference to examples, but the present invention is not limited thereto.