CN111235216A - Method for accurately detecting and killing pathogenic microorganisms by using intelligent bioprobe - Google Patents
Method for accurately detecting and killing pathogenic microorganisms by using intelligent bioprobe Download PDFInfo
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- CN111235216A CN111235216A CN202010119981.7A CN202010119981A CN111235216A CN 111235216 A CN111235216 A CN 111235216A CN 202010119981 A CN202010119981 A CN 202010119981A CN 111235216 A CN111235216 A CN 111235216A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/10—Enterobacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
- A61L2/0029—Radiation
- A61L2/0058—Infrared radiation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/245—Escherichia (G)
Abstract
The invention discloses a method for accurately detecting and killing pathogenic microorganisms by using an intelligent bioprobe, and particularly relates to a programmable intelligent bioprobe which is used for sequentially adding a metal ion precursor solution with a certain concentration and a nucleic acid or target protein solution with a specific structure into a pathogenic microorganism cell suspension, and performing in-situ self-assembly biosynthesis on related pathogenic bacteria or viruses after a period of incubation. The probe can synthesize a fluorescent or magnetic nano probe for accurately targeting and marking pathogenic microorganisms and instantly and quickly detecting pathogenic microorganisms through in-situ biological self-assembly, and can realize quick and accurate tracing and accurate killing of cross-scale multi-mode real-time dynamic high sensitivity and high specificity. The method has the advantages of good biological safety, simplicity, convenience, practicability, high sensitivity, strong specificity, rapid and visual detection and strong universality, and provides a solid technical support for early diagnosis, early warning and monitoring of related diseases caused by highly pathogenic microorganisms.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for instantly, quickly and accurately detecting pathogenic microorganisms based on in-situ self-assembly programmable nucleic acid and other intelligent biological probes.
Background
Pathogenic microorganisms have been "stealth killers" that seriously threaten human health. A reliable, effective and rapid pathogenic microorganism diagnosis method is an important tool for preventing and controlling various diseases (particularly epidemic diseases caused by various coronaviruses) and also an important means for ensuring national and international biological safety. Currently, detection techniques for pathogenic microorganisms include a viable cell count method, a molecular biological method, an enzyme-linked immunosorbent assay method, and the like. The method is complex to operate, the required immunoreagent is expensive and generally has high toxicity, and the problems of false positive detection results or low sensitivity and the like often occur. The biochip is a popular technology developed in recent years, the theoretical basis of which is the nucleic acid hybridization theory, and the biochip is often presented in the form of an array, which has the advantage of realizing multi-channel measurement, but also has the defect of false positive of the measurement result.
With the continuous and intensive biomedical research, the visualized biological imaging technology plays an increasingly important role in the fields of life sciences and medicine. Compared with other biological imaging technologies, the fluorescence imaging technology has the characteristics of low price, fast imaging and the like. Due to the unique physicochemical properties of nano materials such as light, electricity, magnetism and the like, the nano technology has attracted much attention in the aspect of pathogenic microorganism detection in recent years. Metal nanoprobes (e.g., gold, silver, etc.) typically less than 2nm in size, exhibit strong visible-near infrared fluorescence. The synthesis of metal nanoprobes generally requires macromolecular substances such as proteins, nucleic acids, etc. as templates to stabilize the structure of the metal nanoprobes, which is crucial for biological imaging.
DNA and other nucleic acids are used as carriers of genetic codes and can be used as good self-assembly materials due to accurate base complementary pairing. The in vitro self-assembly of nucleic acid and different nano materials can be completed by utilizing the acting force among molecules such as Van der Waals force, covalent bond, electrostatic acting force, charge transfer action and the like. However, these synthesis methods are harsh conditions, such as the need to maintain high temperatures, narrow acid-base ranges, complicated programming, etc. Under the conditions of normal temperature and wide acid-base range, the invention relies on the in-situ self-assembly of pathogenic microorganisms to synthesize fluorescent or magnetic nanoprobes, and directly carries out the marking detection of pathogenic microorganisms, thereby establishing an in-situ self-assembly programmable nucleic acid biomolecule intelligent nanoprobe based on pathogenic bacteria or virus specific microenvironment for the immediate rapid detection and accurate killing of pathogenic microorganisms, and having wide application prospect.
Disclosure of Invention
The technical problem is as follows: aiming at the defects of the prior detection technology, the invention provides an intelligent biological probe for accurately detecting and killing pathogenic microorganisms, which is an intelligent nano probe for in-situ self-assembly of biomolecules such as programmable nucleic acid and the like in a specific microenvironment of pathogenic bacteria or viruses, and establishes a method for instantly and quickly detecting and accurately killing pathogenic microorganisms on the basis of the intelligent nano probe. The method is characterized in that an intelligent biological probe with the characteristics of stable fluorescence or magnetism and the like is synthesized by in-situ self-assembly in a targeted pathogenic microorganism body so as to realize the real-time rapid detection and accurate killing method of pathogenic microorganisms, and the method has the characteristics of high sensitivity, high specificity, simplicity, convenience and the like.
The technical scheme is as follows: the invention discloses an accurate detection method of pathogenic microorganisms of an intelligent biological probe, which is a method for instantly and quickly detecting and accurately killing pathogenic microorganisms of an intelligent nano probe based on biomolecules of in-situ self-assembled programmable nucleic acid of pathogenic bacteria or virus specific microenvironment, and specifically comprises the following steps:
step one, mixing a metal precursor solution with excellent biocompatibility with sterile water to obtain a related metal precursor solution;
secondly, suspending pathogenic microorganism cells by using the related metal precursor solution in the step one, then adding nucleic acid fragments dissolved by sterile water or bioactive substances of a target protein solution, and fully mixing to form a mixed solution A;
step three, placing the mixed liquor A obtained in the step two in a constant temperature shaking table for continuous culture to obtain mixed liquor B;
step four, dripping the mixed solution B processed in the step three on a glass slide for slide preparation;
and fifthly, carrying out fluorescence imaging by utilizing the excitation of a laser confocal fluorescence microscope.
Wherein the content of the first and second substances,
the metal precursor solution with excellent biocompatibility is any one or the combination of any more of a ferrous chloride solution, a chloroauric acid solution, a silver nitrate solution or a zinc gluconate solution.
The nucleic acid fragment comprises any one of a DNA fragment or an RNA fragment or a programmable two-dimensional or three-dimensional structure, or albumin or a targeting antibody and a drug molecule.
And placing the mixed solution in the third step in a constant temperature shaking table for continuous culture for 0.5-12 h.
The pathogenic microorganism killing method of the intelligent bioprobe adopts the mixed solution B prepared in the third step and combines infrared irradiation or other physical intervention to kill pathogenic microorganisms.
Has the advantages that: compared with the prior art, the method has the following advantages and effects:
the invention adopts metal precursor solution, nucleic acid fragments or target protein solution and the like to directly form fluorescent or magnetic metal nano probes with different nucleic acid geometrical structures by in-situ self-assembly in pathogenic microorganisms, realizes accurate target marking and instant rapid detection of pathogenic microorganisms, and has the characteristics of high sensitivity, high specificity, simplicity, convenience and the like. Meanwhile, the fluorescence and magnetic strength and stability of the related intelligent nano probe are enhanced by utilizing the characteristics of the added nucleic acid segment. The method has the advantages of simple operation, immediate and rapid detection, high sensitivity, strong specificity, good universality and the like, and has potential application prospect in the aspects of high-sensitivity rapid diagnosis, immediate detection and the like of pathogenic microorganisms.
Detailed Description
In order to further understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention.
Unless otherwise specified, the reagents and instruments used in the examples of the present invention are commercially available products, and are commercially available.
Step one, mixing a metal precursor solution with excellent biocompatibility with sterile water to obtain a related metal precursor solution;
secondly, suspending pathogenic microorganism cells by using the related metal precursor solution in the step one, then adding nucleic acid fragments dissolved by sterile water or bioactive substances of a target protein solution, and fully mixing to form a mixed solution A;
step three, placing the mixed liquor A obtained in the step two in a constant temperature shaking table for continuous culture to obtain mixed liquor B;
step four, dripping the mixed solution B processed in the step three on a glass slide for slide preparation;
and fifthly, carrying out fluorescence imaging by utilizing the excitation of a laser confocal fluorescence microscope.
Example 1
A method for instantly, quickly and accurately detecting pathogenic microorganisms based on intelligent biological probes such as in-situ self-assembled programmable nucleic acid and the like comprises the following specific steps:
a chloroauric acid solution prepared with sterile water was used as the gold ion precursor solution of the present example, and mixed with E.coli, and then a DNA solution diluted with sterile water at a certain concentration was continuously added as an experimental group. Wherein the final concentration of the chloroauric acid solution is 200 mu mol/L, and the final concentration of the DNA solution is 20 nmol/L. The normal control group and the blank group were set simultaneously. And fully mixing the mixed solution, placing the mixed solution in a constant temperature shaking table, and continuously incubating for 0.5h, 1h or 2-12 h. A small amount of the above mixture was then dropped onto a glass slide and mounted with a coverslip. It was placed under a laser confocal fluorescence microscope for excitation. Further, the mixture was irradiated with 808nm laser light for 10-30min, and cultured and counted. The results showed that E.coli in the experimental group had strong fluorescence phenomenon and was almost completely killed, the normal control group had weak fluorescence and was mostly killed, and the blank group had no change.
Wherein, the normal control group is only added with chloroauric acid solution with the same concentration as the experimental group, and the Escherichia coli suspension of the blank group is not treated.
Example 2
A method for instantly, quickly and accurately detecting pathogenic microorganisms based on intelligent biological probes such as in-situ self-assembled programmable nucleic acid and the like comprises the following specific steps:
a silver nitrate solution prepared with sterile water was used as the silver ion precursor solution of this example, and mixed with escherichia coli, and then a DNA solution diluted with sterile water at a certain concentration was continuously added as an experimental group. Wherein the final concentration of the silver nitrate solution is 200 mu mol/L, and the final concentration of the DNA solution is 20 nmol/L. The normal control group and the blank group were set simultaneously. And fully mixing the mixed solution, placing the mixed solution in a constant temperature shaking table, and continuously incubating for 0.5h, 1h or 2-12 h. A small amount of the above mixture was then dropped onto a glass slide and mounted with a coverslip. It was placed under a laser confocal fluorescence microscope for excitation. Further, the mixture was irradiated with 808nm laser light for 10-30min, and cultured and counted. The results showed that E.coli in the experimental group had strong fluorescence phenomenon and was almost completely killed, the normal control group had weak fluorescence and was mostly killed, and the blank group had no change.
Wherein, the normal control group is only added with silver nitrate solution with the same concentration as the experimental group, and the blank group of escherichia coli suspension is not processed.
Example 3
A method for instantly, quickly and accurately detecting pathogenic microorganisms based on intelligent biological probes such as in-situ self-assembled programmable nucleic acid and the like comprises the following specific steps:
the gold and silver ion precursor solution of this example was prepared with chloroauric acid solution prepared with sterile water and silver nitrate solution, mixed with escherichia coli, and then DNA solution diluted with sterile water at a certain concentration was continuously added as an experimental group. Wherein the final concentration of the chloroauric acid solution and the silver nitrate solution is 200 mu mol/L, and the final concentration of the DNA solution is 20 nmol/L. The normal control group and the blank group were set simultaneously. And fully mixing the mixed solution, placing the mixed solution in a constant temperature shaking table, and continuously incubating for 0.5h, 1h or 2-12 h. A small amount of the above mixture was then dropped onto a glass slide and mounted with a coverslip. It was placed under a laser confocal fluorescence microscope for excitation. Further, the mixture was irradiated with 808nm laser light for 10-30min, and cultured and counted. The results showed that E.coli in the experimental group had strong fluorescence phenomenon and was almost completely killed, the normal control group had weak fluorescence and was mostly killed, and the blank group had no change.
Wherein, the normal control group is only added with chloroauric acid solution and silver nitrate solution with the same concentration as the experimental group, and the escherichia coli suspension of the blank group is not processed.
Example 4
A method for instantly, quickly and accurately detecting pathogenic microorganisms based on intelligent biological probes such as in-situ self-assembled programmable nucleic acid and the like comprises the following specific steps:
the zinc gluconate solution prepared with sterile water was used as the zinc ion precursor solution of this example, and mixed with escherichia coli, and then the DNA solution of a certain concentration diluted with sterile water was continuously added as an experimental group. Wherein the final concentration of the zinc gluconate solution is 200 mu mol/L, and the final concentration of the DNA solution is 20 nmol/L. The normal control group and the blank group were set simultaneously. And fully mixing the mixed solution, placing the mixed solution in a constant temperature shaking table, and continuously incubating for 0.5h, 1h or 2-12 h. A small amount of the above mixture was then dropped onto a glass slide and mounted with a coverslip. It was placed under a laser confocal fluorescence microscope for excitation. Further, the mixture was irradiated with 808nm laser light for 10-30min, and cultured and counted. The results showed that E.coli in the experimental group had strong fluorescence phenomenon and was almost completely killed, the normal control group had weak fluorescence and was mostly killed, and the blank group had no change.
Wherein the normal control group is only added with the zinc gluconate solution with the same concentration as the experimental group, and the blank group of escherichia coli suspension is not treated.
Example 5
A method for instantly, quickly and accurately detecting pathogenic microorganisms based on intelligent biological probes such as in-situ self-assembled programmable nucleic acid and the like comprises the following specific steps:
a ferrous chloride solution and a silver nitrate solution prepared with sterile water were used as the metal ion precursor solution of this example, and mixed with escherichia coli, and then a DNA solution diluted with sterile water at a certain concentration was continuously added as an experimental group. Wherein the final concentration of the ferrous chloride solution and the silver nitrate solution is 200 mu mol/L, and the final concentration of the DNA solution is 20 nmol/L. The normal control group and the blank group were set simultaneously. And fully mixing the mixed solution, placing the mixed solution in a constant temperature shaking table, and continuously incubating for 0.5h, 1h or 2-12 h. A small amount of the above mixture was then dropped onto a glass slide and mounted with a coverslip. It was placed under a laser confocal fluorescence microscope for excitation. Further, the mixture was irradiated with 808nm laser light for 10-30min, and cultured and counted. The results showed that E.coli in the experimental group had strong fluorescence phenomenon and was almost completely killed, the normal control group had weak fluorescence and was mostly killed, and the blank group had no change.
Wherein, the normal control group is only added with ferrous chloride solution and silver nitrate solution with the same concentration as the experimental group, and the Escherichia coli suspension of the blank group is not processed.
While the foregoing is directed to the preferred embodiment of the present invention, it is to be understood that the present invention is illustrative only and is not to be limited in scope by the claims which follow.
Claims (5)
1. A method for accurately detecting pathogenic microorganisms of an intelligent biological probe is characterized in that the detection method is a method for instantly and quickly detecting and accurately killing pathogenic microorganisms of an intelligent nano probe based on biomolecules of in-situ self-assembled programmable nucleic acid of pathogenic bacteria or virus specific microenvironment, and specifically comprises the following steps:
step one, mixing a metal precursor solution with excellent biocompatibility with sterile water to obtain a related metal precursor solution;
secondly, suspending pathogenic microorganism cells by using the related metal precursor solution in the step one, then adding nucleic acid fragments dissolved by sterile water or bioactive substances of a target protein solution, and fully mixing to form a mixed solution A;
step three, placing the mixed liquor A obtained in the step two in a constant temperature shaking table for continuous culture to obtain mixed liquor B;
step four, dripping the mixed solution B processed in the step three on a glass slide for slide preparation;
and fifthly, carrying out fluorescence imaging by utilizing the excitation of a laser confocal fluorescence microscope.
2. The method for accurately detecting pathogenic microorganisms of an intelligent bioprobe according to claim 1, wherein the metal precursor solution with excellent biocompatibility is any one or a combination of any several of ferrous chloride solution, chloroauric acid solution, silver nitrate solution and zinc gluconate solution.
3. The method of claim 1, wherein the nucleic acid fragment comprises any one of a DNA fragment, an RNA fragment, a programmable two-dimensional or three-dimensional structure, or albumin, a targeting antibody, and a drug molecule.
4. The method for accurately detecting pathogenic microorganisms using an intelligent bioprobe as claimed in claim 1, wherein the time for the mixed solution to be continuously cultured on the constant temperature shaking table in step three is 0.5h-12 h.
5. A method for killing pathogenic microorganisms by using an intelligent bioprobe, which is characterized in that the mixed liquor B prepared in the third step of the claim 1 is adopted to kill the pathogenic microorganisms by combining infrared irradiation or other physical interventions.
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| CN114113577A (en) * | 2021-10-22 | 2022-03-01 | 东南大学 | Targeted intelligent probe and preparation method and application thereof |
| WO2022179183A1 (en) * | 2021-02-25 | 2022-09-01 | 东南大学 | Nucleic acid functionalized metal nanoprobe and preparation method therefor |
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