阅读说明：本技术 一种磁性富集材料、水体细菌检测试剂盒及应用 (Magnetic enrichment material, water body bacterium detection kit and application ) 是由 杨燕美 钱玉珍 代莹莹 赵迪 于 2019-11-13 设计创作，主要内容包括：本公开属于细菌检测技术领域,具体涉及一种磁性富集材料、水体细菌检测试剂盒及应用。本公开提供了一种磁性温敏富集材料,采用热敏聚合物聚(N-异丙基异丙烯酰)及刀豆球蛋白A对磁性四氧化三铁纳米颗粒进行修饰,该富集材料通过刀豆球蛋白A对细菌的亲和性实现对水体环境中细菌的吸附作用,通过热敏聚合物的升温相变实现细菌的脱离。本公开还提供了一种包括上述磁性温敏富集材料的试剂盒,还包括对解吸附后的细菌进行显色的试剂。该试剂盒可以实现对水体中细菌的可视化检测,如果颜色溶液发生变化则证明有细菌,并且颜色变化的速度与细菌浓度呈正比。该试剂盒检测速度快,灵敏度高,可应用于水质监测、水污染防治,灾后疫情防控等领域。(The disclosure belongs to the technical field of bacteria detection, and particularly relates to a magnetic enrichment material, a water body bacteria detection kit and application. The magnetic temperature-sensitive enrichment material is characterized in that magnetic ferroferric oxide nanoparticles are modified by adopting a heat-sensitive polymer poly (N-isopropyl isopropenyl) and concanavalin A, the enrichment material realizes the adsorption effect on bacteria in a water body environment through the affinity of the concanavalin A to the bacteria, and the separation of the bacteria is realized through the temperature rise and phase change of the heat-sensitive polymer. The present disclosure also provides a kit comprising the above magnetic temperature-sensitive enrichment material, and further comprises a reagent for developing the color of the desorbed bacteria. The kit can realize the visual detection of bacteria in the water body, if the color solution changes, the bacteria are proved, and the speed of color change is in direct proportion to the concentration of the bacteria. The kit has high detection speed and high sensitivity, and can be applied to the fields of water quality monitoring, water pollution prevention and control, post-disaster epidemic situation prevention and control and the like.)

1. The magnetic temperature-sensitive enrichment nanomaterial is characterized in that the nanomaterial is functionally modified by adopting a heat-sensitive polymer, and bacteria specific molecules are coupled to the surface of the heat-sensitive polymer.

2. The magnetic temperature-sensitive enriched nanomaterial according to claim 1, wherein the thermosensitive polymer is poly (N-isopropylisopropenyl).

3. The magnetic temperature-sensitive enriched nanomaterial according to claim 1, wherein the bacteria-specific molecule is an antibiotic or concanavalin a.

4. The preparation method of the magnetic temperature-sensitive enrichment nano material is characterized by comprising the following steps of: to Fe₃O₄Adding Fe protected by poly (N-isopropyl acrylamide) into magnetic nanoparticles₃O₄And then adding a disuccinimidyl carbonate solution and a sword bean protein solution in sequence to uniformly disperse the mixture to obtain the composition.

5. The use of the magnetic temperature-sensitive enriched nanomaterial of any of claims 1-3 in the preparation of a water body bacteria detection preparation.

6. A water body bacteria detection kit, which is characterized in that the kit comprises the magnetic temperature-sensitive enrichment nano material according to any one of claims 1 to 3.

7. The water body bacteria detection kit of claim 6, wherein the kit further comprises an eluent and a color development liquid; preferably, the eluent is PBS buffer, and the buffer further includes hydrogen phosphate ions and chloride ions.

8. The water body bacteria detection kit of claim 6, wherein the color developing solution comprises a color developing agent C1, a color developing agent C2 and a copper ion-containing reagent; the color developing agent C1 is an alkynyl-modified gold nanoparticle, and the color developing agent C2 is an azide-group-modified gold nanoparticle; preferably, the working temperature of the eluent is-2 to 2 ℃.

9. A method of detecting bacteria in a body of water, the method comprising the steps of:

(1) adding the magnetic temperature-sensitive enrichment nanomaterial as claimed in any one of claims 1 to 3 into a water body to be detected, and oscillating for a period of time to analyze and adsorb bacteria in the water body in the nanomaterial;

(2) removing a liquid part through a magnet solid magnetic temperature-sensitive enrichment material;

(3) adding eluent with working temperature into the magnetic temperature-sensitive enrichment material obtained in the step (2), and oscillating to obtain a liquid part;

(4) adding a color developing agent into the liquid part obtained in the step (3), observing the color change of the liquid part and testing the ultraviolet spectrum.

10. The magnetic temperature-sensitive enriched nanomaterial of any one of claims 1 to 3 and the application of the water body bacteria detection kit of the fourth aspect in the field of water body monitoring.

Technical Field

The disclosure belongs to the technical field of bacteria detection, and particularly relates to a magnetic temperature-sensitive enrichment material, a water body bacteria detection kit comprising the enrichment material, and application of the enrichment material in the fields of water body environment monitoring and water pollution prevention and control.

Background

The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Bacteria are an important pathogen, and after the bacteria invade the body, the bacteria secrete a large amount of biotoxins to destroy the structure and the function of the body, so that host infection is caused, and various diseases such as gastroenteritis, pneumonia, sepsis and the like are caused. In recent years, with the gradual increase of water pollution, infectious diseases caused by pathogenic bacteria in water bodies become important factors which harm human health, and have attracted extensive social attention. Therefore, the research on the rapid analysis and detection technology of the water body bacteria has very important significance for realizing the rapid detection of the water body quality, better guaranteeing the public health of the society and avoiding the economic loss.

The detection techniques for bacteria on the market at present mainly include a bacterial culture method and a Polymerase Chain Reaction (PCR) method. Both methods have their own advantages and disadvantages. The bacterial culture method is the most common analysis method for detecting bacteria at present, but the bacterial culture method has the disadvantages of long time consumption (several days), complex operation, low sensitivity and poor stability, and particularly, the long culture time brings serious inconvenience to the actual water quality detection work. The PCR method has the advantages of high sensitivity, good accuracy and the like, and can simultaneously detect various bacteria. However, the PCR method requires high cost and technical level, is complicated to operate, and requires multiple steps such as bacterial lysis, nucleic acid extraction and amplification. In addition, although the PCR method has greatly shortened the detection period of bacteria (3.5-10 hours) compared to the conventional bacterial culture method, it is difficult to meet the demand of rapid detection in actual fields.

Because the quantity of bacteria in the water body environment is low (usually in a trace amount level of 1-100CFU/mL), if the detection sensitivity of the bacteria can be further improved, the detection work of the actual water body quality can be greatly facilitated.

Disclosure of Invention

Based on the research background, the inventor considers that the establishment of a simple and convenient on-site rapid detection technology for water body bacteria with high sensitivity is very important and urgent. Based on the technical purpose, the method is researched aiming at the enrichment and detection technology of bacteria in the water body, and the method which is higher in adsorption efficiency and more suitable for on-site rapid detection is provided.

Based on the above research background, the present disclosure provides the following technical solutions:

in a first aspect of the disclosure, a magnetic temperature-sensitive enriched nanomaterial is provided, wherein the nanomaterial is functionally modified by a thermosensitive polymer, and a bacteria-specific molecule is coupled to the surface of the thermosensitive polymer.

In the research of Zhuang Men Yao, a canavalin A functionalized magnetic ferroferric oxide nano material is provided, oleic acid is coated on the surface of a nano carrier to protect magnetic ferroferric oxide nano particles, then the surface of the composite carrier is modified through polygamine auto-oxidation, and the composite carrier can perform specific recognition through the canavalin A and mannose components on the surfaces of bacteria, so that the adsorption effect on the bacteria is realized. The disclosure provides an enrichment material with better performance, wherein a thermosensitive polymer poly (N-isopropyl isopropenyl) is adopted as surface modification of magnetic ferroferric oxide, and the polymer can provide more sites for the attachment of the sword bean protein A, thereby greatly improving the bearing capacity of bacteria specific molecules (such as the sword bean protein A) and improving the capture capacity of bacteria; secondly, the polymer is attached to the surface of the magnetic ferroferric oxide, so that the stability of the nano particles can be improved, and the concanavalin A on the surface of the polymer can play a role in protecting the thermosensitive polymer, so that the overall stability is improved.

Preferably, the thermosensitive polymer is poly (N-isopropylisopropenyl).

Preferably, the bacteria-specific molecule is an antibiotic or concanavalin a.

In a second aspect of the disclosure, a magnetic temperature-sensitive material is providedA preparation method of an integrated nano material comprises the following steps: to Fe₃O₄Adding Fe protected by poly (N-isopropyl acrylamide) into magnetic nanoparticles₃O₄And then adding a disuccinimidyl carbonate solution and a sword bean protein solution in sequence to uniformly disperse the mixture to obtain the composition.

Preferably, said Fe₃O₄The preparation method of the magnetic nanoparticles comprises the following steps: mixing Fe₃O₄Dispersing into ethanol, centrifuging to remove supernatant, adding tetramethylammonium hydroxide solution, dispersing again, adding isopropanol, centrifuging to remove supernatant to obtain Fe₃O₄Magnetic nanoparticles.

Preferably, the disuccinimidyl carbonate solution is an acetonitrile solution of N, N' -disuccinimidyl carbonate.

Preferably, the solution of concanavalin is a PBS buffer solution of concanavalin a.

In a third aspect of the disclosure, an application of the magnetic temperature-sensitive enrichment nanomaterial of the first aspect in preparation of a water body bacteria detection preparation is provided.

The enrichment material adopts a magnetic ferroferric oxide nano material, and can be recovered through a magnet after being put into a water body environment; the nanoparticles and the concanavalin A are connected through the thermosensitive polymer, so that the dissociation of bacteria can be conveniently realized by adjusting the temperature of the eluent, and no additional desorption operation is needed. And the surface of the thermosensitive polymer has abundant binding sites for bearing the concanavalin A, so that the capturing capability of bacteria is greatly improved, and the detection sensitivity is improved.

In a fourth aspect of the present disclosure, a water body bacteria detection kit is provided, which includes the magnetic temperature-sensitive enriched nanomaterial of the first aspect.

Preferably, the kit further comprises an eluent and a color development liquid.

Further preferably, the eluent is a PBS buffer, and the buffer further includes hydrogen phosphate ions and chloride ions.

In some specific embodiments, the eluent comprises PBS and Na₂HPO₄、NaCl、KH₂PO₄、KCl。

In some specific embodiments, the eluent is a sterilized reagent.

More preferably, the color developing solution comprises a developer C1, a developer C2 and a copper ion-containing reagent; the color developing agent C1 is alkynyl (SH-ALK) modified gold nanoparticles, and the color developing agent C2 is azide group (SH-N)₃) Modified gold nanoparticles.

The gold nanoparticles are respectively subjected to alkynyl and azide group functional modification, and Cu is reduced by bacteria²⁺And a click reaction is initiated, so that cross-linking and agglomeration are caused among the gold nanoparticles modified by the alkynyl group and the azide group, and high-specificity on-site rapid detection of water body bacteria is realized through the color change (from red to grey) of the nano-gold solution.

Further preferably, the working temperature of the eluent is-2 to 2 ℃.

The kit provided by the disclosure captures bacteria in a water body environment through the enrichment material, and realizes the separation of the bacteria and the nano material by adjusting the temperature of the eluent, and the temperature of the eluent when added into the liquid to be detected, namely the working temperature, is preferably-2 ℃, and the polymer can be separated from the surface of the magnetic nano material through the swelling action in a low-temperature state to realize the desorption of the bacteria, wherein the optimum working temperature is 0 ℃.

In a fifth aspect of the present disclosure, there is provided a method for detecting bacteria in a body of water, the method comprising the steps of:

(1) adding the magnetic temperature-sensitive enrichment nanomaterial of the first aspect into a water body to be detected, and oscillating for a period of time to analyze and adsorb bacteria in the water body in the nanomaterial;

(2) removing a liquid part through a magnet solid magnetic temperature-sensitive enrichment material;

(3) adding eluent with working temperature into the magnetic temperature-sensitive enrichment material obtained in the step (2), and oscillating to obtain a liquid part;

(4) adding a color developing agent into the liquid part obtained in the step (3), observing the color change of the liquid part and testing the ultraviolet spectrum.

The disclosure provides, in a sixth aspect, an application of the magnetic temperature-sensitive enriched nanomaterial of the first aspect and the water body bacteria detection kit of the fourth aspect in the field of water body monitoring.

Compared with the prior art, the beneficial effect of this disclosure is:

1. the adsorption material with higher enrichment efficiency is provided by taking a magnetic nano material as a carrier, can conveniently realize the adsorption and the separation of bacteria through a thermosensitive polymer, and provides more abundant binding sites for bacteria adsorption factors.

2. The present disclosure also provides a kit for detecting bacteria in a water body, wherein the kit comprises an enrichment material, an eluent and a color development liquid. Based on above-mentioned magnetism temperature sensitive enrichment nano-material, this disclosure uses magnetism nano-material as the carrier, can carry out convenient fixed with nano-material through magnet to can realize the quick desorption of bacterium through the operating temperature of adjustment eluant, the concentration of bacterium in the water can conveniently be confirmed through the color change of visual observation reagent, the detection requirement that is particularly useful for the on-the-spot official working.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 is a schematic diagram of rapid detection of water bacteria based on a magnetic temperature-controlled enriched bacteria nano reagent and a click reaction assisted nano-gold colorimetric method in the present disclosure;

FIG. 2 is a color change diagram of the bacterial liquid of example 3 after the addition of a color-developing agent;

wherein FIG. 2A shows the solution in red without the addition of copper sulfate solution;

FIG. 2B shows the solution changing from red to blue 30s after the addition of the copper sulfate solution;

FIG. 2C shows that 30min after the addition of copper sulfate solution, the solution gradually turns dark blue;

FIG. 2D shows that the solution became colorless 3h after the addition of the copper sulfate solution.

FIG. 3 is a UV spectrum of Escherichia coli and Staphylococcus aureus in example 3;

wherein, FIG. 3A shows the concentration of 10⁵Ultraviolet spectrum of the escherichia coli eluent;

FIG. 3B shows the concentration of 10⁷Ultraviolet spectrum of Escherichia coli;

FIG. 3C shows the concentration of 10⁵An ultraviolet spectrum of the staphylococcus aureus eluent;

FIG. 3D shows the concentration of 10⁷(ii) a staphylococcus aureus ultraviolet spectrum;

FIG. 4 is a sample diagram of the kit described in example 2;

from left to right, ferroferric oxide nanoparticle solution, PBS eluent, SH-ALK modified gold nanoparticle solution, SH-N3 modified gold nanoparticle solution and CuSO are sequentially added₄The solution is black, colorless, red, purple red and blue in sequence.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As introduced in the background art, in order to overcome the defects in the prior art, the present disclosure provides a magnetic temperature-sensitive enrichment nanomaterial and a kit for detecting bacteria in a water body.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.