CN118518872B - Fungus detection method and device - Google Patents
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- 241000233866 Fungi Species 0.000 title claims abstract description 19
- 241000222122 Candida albicans Species 0.000 claims abstract description 82
- 229940095731 candida albicans Drugs 0.000 claims abstract description 82
- 239000002077 nanosphere Substances 0.000 claims abstract description 45
- 238000006243 chemical reaction Methods 0.000 claims abstract description 31
- 229910001112 rose gold Inorganic materials 0.000 claims abstract description 23
- 238000003908 quality control method Methods 0.000 claims abstract description 21
- 238000005507 spraying Methods 0.000 claims abstract description 20
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 18
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 57
- 241000228212 Aspergillus Species 0.000 claims description 51
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- 239000006228 supernatant Substances 0.000 claims description 35
- 239000012528 membrane Substances 0.000 claims description 31
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- 239000012279 sodium borohydride Substances 0.000 claims description 23
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 20
- 239000002202 Polyethylene glycol Substances 0.000 claims description 20
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- 238000002156 mixing Methods 0.000 claims description 18
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 15
- 229940038773 trisodium citrate Drugs 0.000 claims description 15
- DLNWMWYCSOQYSQ-UHFFFAOYSA-M benzyl-hexadecyl-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 DLNWMWYCSOQYSQ-UHFFFAOYSA-M 0.000 claims description 14
- IZWSFJTYBVKZNK-UHFFFAOYSA-N lauryl sulfobetaine Chemical compound CCCCCCCCCCCC[N+](C)(C)CCCS([O-])(=O)=O IZWSFJTYBVKZNK-UHFFFAOYSA-N 0.000 claims description 14
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 13
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- 229930006000 Sucrose Natural products 0.000 claims description 11
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 11
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- JAJIPIAHCFBEPI-UHFFFAOYSA-N 9,10-dioxoanthracene-1-sulfonic acid Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C=CC=C2S(=O)(=O)O JAJIPIAHCFBEPI-UHFFFAOYSA-N 0.000 claims 1
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- SDKPSXWGRWWLKR-UHFFFAOYSA-M sodium;9,10-dioxoanthracene-1-sulfonate Chemical compound [Na+].O=C1C2=CC=CC=C2C(=O)C2=C1C=CC=C2S(=O)(=O)[O-] SDKPSXWGRWWLKR-UHFFFAOYSA-M 0.000 description 3
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- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
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- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
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- 241000202934 Mycoplasma pneumoniae Species 0.000 description 1
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56961—Plant cells or fungi
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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- G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
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- G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
- G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida
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Abstract
The invention provides a fungus detection method and device, and relates to the technical field of detection. The detection method comprises the steps of combining candida albicans antibodies on red gold nanospheres to form candida albicans antibody marked red gold nanosphere solution, and spraying the candida albicans antibody marked red gold nanosphere solution on a combining pad; spraying and coating candida albicans antibodies and rabbit anti-sheep IgG on the reaction pad to form a first detection line and a quality control line which can be developed; the sample dropped on the sample pad moves to the first detection line through the binding pad, and can be combined with the candida albicans antibody on the first detection line to display corresponding color, so that whether candida albicans is contained in the sample can be detected rapidly, and meanwhile, false positive cannot occur.
Description
Technical Field
The invention relates to the technical field of fungus detection, in particular to a fungus detection method and a fungus detection device.
Background
The detection of common fungi such as candida albicans, aspergillus, cryptococcus and the like takes colloidal gold as a color development marker. The colloidal gold particles are negatively charged in a weak alkaline environment, can be electrostatically combined with positively charged genes such as protein molecules and the like, and do not influence biological characteristics of the colloidal gold particles, so that the colloidal gold particles can be specifically combined with an object to be detected (antibody, antigen, small molecule and the like), and the corresponding molecules to be detected are determined. The detection time of the colloidal gold detection method generally shows the detection result in a longer time, and the false positive condition is higher. At present, the prior art cannot realize rapid and accurate type determination on candida albicans, aspergillus and cryptococcus.
Disclosure of Invention
The invention aims to provide a fungus detection method and a fungus detection device, which are used for solving the technical problems of low fungus type determination speed and poor reliability.
In order to achieve the above object, the present invention provides the following technical solutions:
in a first aspect, the present invention provides a method of detecting a fungus, including candida albicans, comprising:
s1.1, adding gold chloride and trisodium citrate into deionized water, heating and stirring in a magnetic stirring heater to obtain a gold chloride and trisodium citrate mixed solution, wherein the mass ratio of the gold chloride to the trisodium citrate is 4:1; adding sodium borohydride to deionized water until dissolved in an ice bath environment to obtain a sodium borohydride solution of 4 mg/ml; adding sodium borohydride solution into the mixed solution of gold chloride and trisodium citrate, stirring, cooling at room temperature, and standing in a dark environment to turn into red to obtain red color nanosphere solution; wherein the volume of the sodium borohydride solution is 0.25% of the volume of the mixed solution of gold chloride and trisodium citrate;
S1.2, adding candida albicans antibody into pure water and PBS buffer solution, centrifuging and taking supernatant to prepare candida albicans antibody solution with the concentration of 6 mg/ml;
S1.3, after the pH value of the red color nanosphere solution is regulated to 9.2, adding the candida albicans antibody solution, mixing and stirring, adding bovine serum albumin with the mass concentration of 10% and polyethylene glycol with the mass concentration of 0.2%, and centrifuging to obtain supernatant; adding a mixed solution of sucrose with the mass concentration of 15% and boric acid with the mass concentration of 0.02% into the supernatant, and obtaining a candida albicans antibody marked red-colored nanosphere solution after volume fixing;
S1.4, spraying the candida albicans antibody marked red color nano ball solution on a glass fiber film to form a bonding pad;
S1.5, respectively diluting candida albicans antibodies and rabbit anti-sheep IgG with PBS buffer solution to a concentration of 3mg/ml, and respectively spraying the candida albicans antibodies and the rabbit anti-sheep IgG on a modified nitrocellulose membrane to be embedded on a reaction pad to form a first detection line and a quality control line;
S1.6, dripping a sample to be detected to a sample pad, moving the sample to a quality control line and a first detection line of the reaction pad through the combination pad, and determining whether the sample contains candida albicans through color development of the first detection line.
In a second aspect, the present invention also provides a detection device, applied to the detection method of the first aspect, the detection device comprising a substrate, and a sample pad, a binding pad, a reaction pad and a water absorbing pad provided on the substrate, the sample pad having a detection hole for receiving a sample;
One end of the bonding pad is laminated with one end of the sample pad, and the other end of the bonding pad is laminated with one end of the reaction pad;
One end of the water absorption pad is laminated with one end of the reaction pad, which is away from the bonding pad.
In one or more of the technical solutions provided in the exemplary embodiments of the present invention, at least one of the following advantages may be achieved.
According to the fungus detection method of the exemplary embodiment of the invention, candida albicans antibodies are combined on red gold nanospheres to form candida albicans antibody marked red gold nanosphere solution, and the candida albicans antibody marked red gold nanosphere solution is sprayed on a combining pad; spraying and coating candida albicans antibodies and rabbit anti-sheep IgG on the reaction pad to form a first detection line and a quality control line which can be developed; through the absorption of the reaction pad, the sample dropped on the sample pad moves to the first detection line through the combination pad, can be combined with the candida albicans antibody on the first detection line so as to display the color of the red nanospheres, and further can rapidly detect whether the sample contains candida albicans or not, and meanwhile, false positive can not occur.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention;
FIG. 1 is a schematic flow chart of a detection method according to an embodiment of the invention;
Fig. 2 is a schematic diagram of an axial structure of a detection device according to an embodiment of the present invention.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In addition, embodiments of the present disclosure and features of the embodiments may be combined with each other without conflict. The present disclosure will be described in detail below with reference to the accompanying drawings in conjunction with embodiments. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Candida albicans antibodies were purchased from zhuhai bomei biotechnology limited; aspergillus and Cryptococcus antibodies were purchased from Dana (Tianjin) Biotechnology Inc.
Fig. 1 is a schematic flow chart of a detection method according to an embodiment of the present invention. As shown in fig. 1, the detection method of candida albicans, aspergillus and cryptococcus provided by the exemplary embodiment of the invention comprises the following steps:
s101, adding 4mg/ml sodium borohydride solution into a gold chloride and trisodium citrate mixed solution, and standing in a dark environment to change into red to obtain a red color nanosphere solution; wherein the mass ratio of the gold chloride to the trisodium citrate is 4:1.
Step S102, mixing a first mixed aqueous solution of lauryl sulfobetaine and benzyl dimethyl hexadecyl ammonium bromide with a gold salt solution of gold chloride, sequentially adding a silver nitrate solution, an L-ascorbic acid solution and a red gold nanosphere solution, and standing in a dark environment to turn blue to obtain a blue-colored nanostar solution; wherein the mass ratio of the lauryl sulfobetaine to the benzyl dimethyl hexadecyl ammonium bromide to the gold chloride to the silver nitrate to the L-ascorbic acid is 15:10:10:1:9.
The above-described laurylsulfobetaine and benzyldimethyl hexadecyl ammonium bromide allow for the adjustment of the size and branching degree of gold nanosatellites such that the size of gold nanosatellites is stable at high salt concentrations and has sufficient branching to aggregate around aspergillus causing a color change.
Step S103, adding a blue-colored nanostar solution into a second mixed aqueous solution of lauryl sulfobetaine, benzyl dimethyl hexadecyl ammonium bromide and phthalic dithiol, mixing, adding nano silver particles, performing ultrasonic dispersion, and standing to obtain a black gold nanostar solution with silver at the tip; wherein the mass ratio of the lauryl sulfobetaine to the benzyl dimethyl hexadecyl ammonium bromide to the phthalic dithiol to the nano silver particles is 15:10:5:1.
And step S104, adding the bacterial antibody into pure water and PBS buffer solution, centrifuging, and taking supernatant to prepare 6mg/ml bacterial antibody solution.
Step S105, after the PH value of the nanoparticle solution corresponding to the bacterial antibody is regulated to 9.2, adding the bacterial antibody solution, mixing and stirring, adding bovine serum albumin with the mass concentration of 10% and polyethylene glycol with the mass concentration of 0.2%, and centrifuging to obtain supernatant; adding a mixed solution of sucrose with the mass concentration of 15% and boric acid with the mass concentration of 0.02% into the supernatant, and obtaining the bacterial antibody marked nanoparticle solution after volume fixing.
In step S104 and step S105, the bacterial antibodies include one of candida albicans antibodies, aspergillus antibodies and cryptococcus antibodies; the nanoparticle solution is one of a red gold nanosphere solution corresponding to candida albicans antibody, a blue gold nanostar solution corresponding to aspergillus antibody and a black gold nanostar solution with silver at the tip corresponding to cryptococcus antibody; the fungus antibody marked nanoparticle solution comprises candida albicans antibody marked red-colored nanosphere solution, aspergillus antibody marked blue-colored nanostar solution and cryptococcus antibody marked black-end silver-bearing gold nanostar solution.
And (5) repeating the step S104 and the step S105 to prepare candida albicans antibody marked red-colored nanosphere solution, aspergillus antibody marked blue-colored nanostar solution and cryptococcus antibody marked black gold nanostar solution with silver on the tip.
Step S106, spraying the candida albicans antibody marked red-colored nanosphere solution, the aspergillus antibody marked blue-colored nanostar solution and the cryptococcus antibody marked black gold nanostar solution with silver at the tip on a glass fiber film to form a bonding pad; the concentration of the bacteria antibody marked nanoparticle solution reaches 0.5-3mg/ml based on the bacteria antibody, so that an effective binding pad can be formed.
And S107, respectively diluting candida albicans antibodies, cryptococcus antibodies, aspergillus antibodies and rabbit anti-sheep IgG with PBS buffer solution until the concentration is 3mg/ml, and respectively spraying the candida albicans antibodies, the cryptococcus antibodies, the aspergillus antibodies and the rabbit anti-sheep IgG on a modified nitrocellulose membrane coating to form a plurality of detection lines and quality control lines.
And S108, dripping the sample to be detected to a sample pad, moving the sample to a quality control line and each detection line of the reaction pad through a combination pad, and determining the type of the sample through the color development of each detection line.
In an alternative embodiment, in step S1.1, the obtained red gold nanosphere solution is stored at a low temperature for standby after being sealed.
In an alternative embodiment, in step S3.1, the volume ratio of the second mixed aqueous solution to the blue nano-star solution is 25:3.
In an alternative embodiment, in step S1.3, a potassium carbonate solution with a concentration of 0.3mol/L is added to the red gold nanosphere solution to adjust the pH.
In an alternative embodiment, in step S1.3, after adding the candida albicans antibody solution, mixing and stirring for 30min, adding bovine serum albumin and polyethylene glycol sequentially under stirring, continuing stirring for 40min, and centrifuging to obtain a supernatant.
In an alternative embodiment, after forming the conjugate pad in step S1.4, the conjugate pad is vacuum freeze-dried for 24h to 36h.
In an alternative embodiment, in step S1.5, the preparation of the modified nitrocellulose membrane comprises: the nitrocellulose membrane is soaked in a treatment liquid and is cleaned by distilled water after oscillation, wherein the treatment liquid consists of 0.2% by mass of L-phenylalanine, 0.3% by mass of polyethylene glycol and water.
Fig. 2 is a schematic diagram of an axial structure of a detection device according to an embodiment of the present invention. As shown in fig. 2, the exemplary embodiment of the present invention also provides a fungus detection device, which is applied to the above detection method, the detection device includes a substrate 50, and a sample pad 10, a binding pad 20, a reaction pad 30, and a water absorbing pad 40 disposed on the substrate 50, the sample pad 10 having a detection hole 11 for receiving a sample; one end of the bonding pad 20 is laminated with one end of the sample pad 10, and the other end is laminated with one end of the reaction pad 30; one end of the water absorbing pad 40 is laminated with one end of the reaction pad 30 facing away from the bonding pad 20.
The reaction pad 30 is provided with a first detection line 31 formed by pre-coating candida albicans antibodies on a modified nitrocellulose membrane, a second detection line 32 pre-coating aspergillus antibodies on the modified nitrocellulose membrane, a third detection line 33 pre-coating cryptococcus antibodies on the modified nitrocellulose membrane, and a quality control line 34 pre-coating rabbit anti-sheep IgG on the modified nitrocellulose membrane.
In actual detection, 100. Mu.L of a sample to be detected is dripped into the detection hole 11 at room temperature, and whether the sample contains candida albicans, aspergillus and cryptococcus is determined by the color development of the first detection line 31, the second detection line 32, the third detection line 33 and the quality control line 34. The water absorbing pad 40 serves to move the sample dropped on the sample pad 10 to the reaction pad 30 through the binding pad 20.
Illustratively, only candida albicans antibody labeled red color nanosphere solution is sprayed on the bonding pad 20, and only the first detection line 31 is arranged on the reaction pad 30 to detect only candida albicans; only spraying aspergillus antibody marked blue-colored nanostar solution on the bonding pad 20, and only setting a second detection line 32 on the reaction pad 30 to detect aspergillus only; only cryptococcus antibody is sprayed on the bonding pad 20 to mark the gold nano-star solution with silver at the black tip, and only the third detection line 33 is arranged on the reaction pad 30 to detect only cryptococcus. The three bacteria antibodies can be sprayed on the binding pad 20 according to actual needs to mark nano particles, and the corresponding three detection lines are arranged on the reaction pad 30, so as to realize detection of candida albicans, aspergillus and cryptococcus at the same time.
The detection results include the following classifications:
Positive: if the sample contains candida albicans, aspergillus and cryptococcus, the corresponding color appears in each detection line, and the quality control line 34 appears red; for example: the first detection line 31 shows that the red representative sample contains candida albicans; the second detection line 32 shows that blue represents that the sample contains aspergillus; the third detection line 33 shows that the black color represents that cryptococcus is contained in the sample.
Negative: no color appears in each detection line, and the quality control line 34 appears red;
Failure: no corresponding color appears in each of the detection lines and the quality control line 34.
Example 1
S1, preparing a red gold nanosphere solution:
(1) Putting 20mL of deionized water into a siliconizing bottle, adding 2mg of gold chloride and 0.5mg of trisodium citrate, heating to boiling in a magnetic stirring heater, and stirring for 10 minutes to obtain a solution;
(2) The vessel was placed in an ice bath, 1mL of deionized water was added to the vessel, and 4mg of sodium borohydride was added to the above vessel and kept cool until the sodium borohydride was dissolved to prepare a sodium borohydride solution,
(3) Adding 50 mu L of sodium borohydride solution into the solution in the step (1) under vigorous stirring, cooling the solution at room temperature after stirring for 5 minutes, standing for 12 hours in a dark environment, changing the solution into red, filtering the solution by a filter, namely preparing a red color nanosphere solution with the particle size of 15nm, sealing the solution, and placing the sealed solution in a refrigerator with the temperature of 4 ℃ for later use.
S4.1, preparing a candida albicans antibody marked red-colored nanosphere solution:
(1) Adding candida albicans antibody into 200 mu L of pure water and 100 mu L of buffer PBS, centrifuging to obtain supernatant, and obtaining candida albicans antibody solution with the concentration of 6 mg/ml;
(2) Taking 8mL of the red gold nanosphere solution prepared in the step S1, adding 60 mu L of potassium carbonate solution with the concentration of 0.3mol/L, adjusting the pH value to 9.2, then mixing and stirring with the candida albicans antibody solution in the step (1) for 30min to enable candida albicans antibodies to be fully combined on the red gold nanospheres, then sequentially adding 8mL of bovine serum albumin with the mass concentration of 10% and 8mL of 0.2% polyethylene glycol under the stirring state, stirring for 40min, and centrifuging to obtain supernatant;
(3) Adding a mixed solution of sucrose with a mass concentration of 15% and boric acid with a mass concentration of 0.02% to the supernatant of the step (2) to a volume of 900. Mu.L.
And S5, spraying the candida albicans antibody marked red color nanosphere solution prepared in the step S4.1 onto a glass fiber membrane to form a bonding pad, and performing vacuum freeze drying for 24 hours.
S6.1, soaking the nitrocellulose membrane in the treatment liquid for 50min, shaking slowly, taking out, cleaning with distilled water for 3 times, and finally drying in a vacuum drying oven. The treatment fluid consists of 0.2% L-phenylalanine, 0.3% polyethylene glycol and water.
S6.2, respectively diluting candida albicans antibodies and rabbit anti-sheep IgG with PBS buffer solution until the concentration is 3mg/ml, respectively spraying the candida albicans antibodies and the rabbit anti-sheep IgG on a modified nitrocellulose membrane, and pre-coating candida albicans antibodies (a first detection line 31) and rabbit anti-sheep IgG (a quality control line 34) on the modified nitrocellulose membrane to form a reaction pad 30.
S7, 100 mu L of a sample to be detected is dripped into the detection hole 11 at room temperature, and whether the sample contains candida albicans is determined through the color development of the first detection line 31 and the quality control line 34.
Example 2
S1, preparing a red gold nanosphere solution:
(1) Putting 20mL of deionized water into a siliconizing bottle, adding 2mg of gold chloride and 0.5mg of trisodium citrate, heating to boiling in a magnetic stirring heater, and stirring for 10 minutes to obtain a solution;
(2) The vessel was placed in an ice bath, 1mL of deionized water was added to the vessel, and 4mg of sodium borohydride was added to the above vessel and kept cool until the sodium borohydride was dissolved to prepare a sodium borohydride solution,
(3) Adding 50 mu L of sodium borohydride solution into the solution in the step (1) under vigorous stirring, cooling the solution at room temperature after stirring for 5 minutes, standing for 12 hours in a dark environment, changing the solution into red, filtering the solution by a filter, namely preparing a red color nanosphere solution with the particle size of 15nm, sealing the solution, and placing the sealed solution in a refrigerator with the temperature of 4 ℃ for later use.
S2, preparing a blue-color nano star solution:
(1) To 50ml of deionized water was added 30mg of lauryl sulfobetaine and 20mg of benzyl dimethyl hexadecyl ammonium bromide to prepare a solution;
(2) Adding 20mg of gold chloride into 5ml of deionized water to prepare a gold salt solution;
(3) Adding 2mg of silver nitrate into 1ml of deionized water to prepare a silver nitrate solution;
(4) 18mg of L-ascorbic acid was added to 1ml of deionized water to prepare an L-ascorbic acid solution;
(5) Mixing the solutions in the step (1) and the step (2), adding the silver nitrate solution in the step (3), then dropwise adding the L-ascorbic acid solution in the step (4), then adding 1ml of the red spherical gold nanoparticle solution in the step (S1), changing the color of the solution into blue, namely finally generating a blue gold nanoparticle solution with the particle size of 30nm, and preserving in a dark environment at room temperature.
S4.1, preparing a candida albicans antibody marked red-colored nanosphere solution:
(1) Adding candida albicans antibody into 200 mu L of pure water and 100 mu L of buffer PBS, centrifuging to obtain supernatant, and obtaining candida albicans antibody solution with the concentration of 6 mg/ml;
(2) Taking 8mL of the red gold nanosphere solution prepared in the step S1, adding 60 mu L of potassium carbonate solution with the concentration of 0.3mol/L, adjusting the pH value to 9.2, then mixing and stirring with the candida albicans antibody solution in the step (1) for 30min to enable candida albicans antibodies to be fully combined on the red gold nanospheres, then sequentially adding 8mL of bovine serum albumin with the mass concentration of 10% and 8mL of 0.2% polyethylene glycol under the stirring state, stirring for 40min, and centrifuging to obtain supernatant;
(3) Adding a mixed solution of sucrose with a mass concentration of 15% and boric acid with a mass concentration of 0.02% to the supernatant of the step (2) to a volume of 900. Mu.L.
S4.2, preparation of an aspergillus antibody marked blue-color nano star solution:
(1) Adding the aspergillus antibody into 200 mu L of pure water and 100 mu L of buffer PBS, centrifuging and taking the supernatant to obtain an aspergillus antibody solution with the concentration of 6 mg/ml;
(2) Taking 8mL of the blue-colored nano-star solution prepared in the step S2, adding 60 mu L of potassium carbonate solution with the concentration of 0.3mol/L, adjusting the pH value to 9.2, then mixing and stirring with the aspergillus antibody solution in the step (1) for 30min to enable the aspergillus antibody to be fully combined with the blue-colored nano-star, then sequentially adding 8mL of 10% bovine serum albumin and 8mL of 0.2% polyethylene glycol under the stirring state, stirring for 40min, and centrifuging to obtain supernatant;
(3) Adding a mixed solution of sucrose with a mass concentration of 15% and boric acid with a mass concentration of 0.02% to the supernatant of the step (2) to a volume of 900. Mu.L.
S5, spraying the candida albicans antibody marked red-colored nanosphere solution and the aspergillus antibody marked blue-colored nanosphere solution prepared in the steps S4.1-S4.2 on a glass fiber membrane to form a bonding pad, and performing vacuum freeze drying for 24 hours.
S6.1, soaking the nitrocellulose membrane in the treatment liquid for 50min, shaking slowly, taking out, cleaning with distilled water for 3 times, and finally drying in a vacuum drying oven. The treatment fluid consists of 0.2% L-phenylalanine, 0.3% polyethylene glycol and water.
S6.2, respectively diluting candida albicans antibody, aspergillus antibody and rabbit anti-sheep IgG with PBS buffer solution until the concentration is 3mg/ml, respectively spraying the candida albicans antibody, the aspergillus antibody and the rabbit anti-sheep IgG on a modified nitrocellulose membrane, and pre-coating candida albicans antibody (a first detection line 31), aspergillus antibody (a second detection line 32) and rabbit anti-sheep IgG (a quality control line 34) on the modified nitrocellulose membrane to form a reaction pad 30.
S7, 100 mu L of a sample to be detected is dripped into the detection hole 11 at room temperature, and whether the sample contains candida albicans and aspergillus is determined through the color development of the first detection line 31, the second detection line 32 and the quality control line 34.
Example 3
S1, preparing a red gold nanosphere solution:
(1) Putting 20mL of deionized water into a siliconizing bottle, adding 2mg of gold chloride and 0.5mg of trisodium citrate, heating to boiling in a magnetic stirring heater, and stirring for 10 minutes to obtain a solution;
(2) The vessel was placed in an ice bath, 1mL of deionized water was added to the vessel, and 4mg of sodium borohydride was added to the above vessel and kept cool until the sodium borohydride was dissolved to prepare a sodium borohydride solution,
(3) Adding 50 mu L of sodium borohydride solution into the solution in the step (1) under vigorous stirring, cooling the solution at room temperature after stirring for 5 minutes, standing for 12 hours in a dark environment, changing the solution into red, filtering the solution by a filter, namely preparing a red color nanosphere solution with the particle size of 15nm, sealing the solution, and placing the sealed solution in a refrigerator with the temperature of 4 ℃ for later use.
S2, preparing a blue-color nano star solution:
(1) To 50ml of deionized water was added 30mg of lauryl sulfobetaine and 20mg of benzyl dimethyl hexadecyl ammonium bromide to prepare a solution;
(2) Adding 20mg of gold chloride into 5ml of deionized water to prepare a gold salt solution;
(3) Adding 2mg of silver nitrate into 1ml of deionized water to prepare a silver nitrate solution;
(4) 18mg of L-ascorbic acid was added to 1ml of deionized water to prepare an L-ascorbic acid solution;
(5) Mixing the solutions in the step (1) and the step (2), adding the silver nitrate solution in the step (3), then dropwise adding the L-ascorbic acid solution in the step (4), then adding 1ml of the red spherical gold nanoparticle solution in the step (S1), changing the color of the solution into blue, namely finally generating a blue gold nanoparticle solution with the particle size of 30nm, and preserving in a dark environment at room temperature.
S3, preparing a gold nanoscin solution with silver at the black terminal:
(1) To 50ml of deionized water was added 30mg of lauryl sulfobetaine and 20mg of benzyl dimethyl hexadecyl ammonium bromide and 10mg of phthalic dithiol to prepare a solution;
(2) Adding 6ml of the blue-colored nano-star solution prepared in the step S2 into the solution in the step (1) to prepare a mixed solution;
(3) Adding 2mg of nano silver particles into the mixed solution in the step (2), and after ultrasonic dispersion for 30min, attaching the silver nano particles to the tips of the gold nano-star through phthalic dithiol to finally form a gold nano-star solution with 40nm black tips and silver.
S4.1, preparing a candida albicans antibody marked red-colored nanosphere solution:
(1) Adding candida albicans antibody into 200 mu L of pure water and 100 mu L of buffer PBS, centrifuging to obtain supernatant, and obtaining candida albicans antibody solution with the concentration of 6 mg/ml;
(2) Taking 8mL of the red gold nanosphere solution prepared in the step S1, adding 60 mu L of potassium carbonate solution with the concentration of 0.3mol/L, adjusting the pH value to 9.2, then mixing and stirring with the candida albicans antibody solution in the step (1) for 30min to enable candida albicans antibodies to be fully combined on the red gold nanospheres, then sequentially adding 8mL of bovine serum albumin with the mass concentration of 10% and 8mL of 0.2% polyethylene glycol under the stirring state, stirring for 40min, and centrifuging to obtain supernatant;
(3) Adding a mixed solution of sucrose with a mass concentration of 15% and boric acid with a mass concentration of 0.02% to the supernatant of the step (2) to a volume of 900. Mu.L.
S4.2, preparation of an aspergillus antibody marked blue-color nano star solution:
(1) Adding the aspergillus antibody into 200 mu L of pure water and 100 mu L of buffer PBS, centrifuging and taking the supernatant to obtain an aspergillus antibody solution with the concentration of 6 mg/ml;
(2) Taking 8mL of the blue-colored nano-star solution prepared in the step S2, adding 60 mu L of potassium carbonate solution with the concentration of 0.3mol/L, adjusting the pH value to 9.2, then mixing and stirring with the aspergillus antibody solution in the step (1) for 30min to enable the aspergillus antibody to be fully combined with the blue-colored nano-star, then sequentially adding 8mL of bovine serum albumin with the mass concentration of 10% and 8mL of 0.2% polyethylene glycol under the stirring state, stirring for 40min, and centrifuging to obtain supernatant;
(3) Adding a mixed solution of sucrose with a mass concentration of 15% and boric acid with a mass concentration of 0.02% to the supernatant of the step (2) to a volume of 900. Mu.L.
S4.3, preparing a gold nanoscin solution with black tips marked with cryptococcus antibody and silver:
(1) Adding cryptococcus antibody into 200 mu L of pure water and 100 mu L of buffer PBS, centrifuging to obtain supernatant, and obtaining cryptococcus antibody solution with the concentration of 6 mg/ml;
(2) Taking 8mL of the gold nanoscin solution with silver at the black terminal, which is prepared in the step S3, adding 60 mu L of potassium carbonate solution with the concentration of 0.3mol/L, adjusting the pH value to 9.2, then mixing and stirring the solution with the cryptococcus antibody solution in the step (1) for 30min, fully combining the cryptococcus antibody with the gold nanoscin with silver at the black terminal, then sequentially adding 8mL of 10% bovine serum albumin and 8mL of 0.2% polyethylene glycol under the stirring state, stirring for 40min, and centrifuging to obtain supernatant;
(3) Adding a mixed solution of sucrose with a mass concentration of 15% and boric acid with a mass concentration of 0.02% to the supernatant of the step (2) to a volume of 900. Mu.L.
S5, spraying the candida albicans antibody marked red-colored nanosphere solution, the aspergillus antibody marked blue-colored nanostar solution and the cryptococcus antibody marked black gold nanostar solution with silver at the peripheral ends prepared in the steps S4.1-S4.3 onto a glass fiber membrane to form a bonding pad, and performing vacuum freeze drying for 24 hours.
S6.1, soaking the nitrocellulose membrane in the treatment liquid for 50min, shaking slowly, taking out, cleaning with distilled water for 3 times, and finally drying in a vacuum drying oven. The treatment fluid consists of 0.2% L-phenylalanine, 0.3% polyethylene glycol and water.
S6.2, respectively diluting candida albicans antibodies, aspergillus antibodies, cryptococcus antibodies and rabbit anti-sheep IgG with PBS buffer solution until the concentration is 3mg/ml, respectively spraying the diluted candida albicans antibodies, aspergillus antibodies, cryptococcus antibodies and rabbit anti-sheep IgG on a modified nitrocellulose membrane, and forming a reaction pad 30 by pre-coating candida albicans antibodies (a first detection line 31), aspergillus antibodies (a second detection line 32), cryptococcus antibodies (a third detection line 33) and rabbit anti-sheep IgG (a quality control line 34) on the modified nitrocellulose membrane.
S7, 100 mu L of a sample to be detected is dripped into the detection hole 11 at room temperature, and whether the sample contains candida albicans, aspergillus and cryptococcus is determined through the color development of the first detection line 31, the second detection line 32, the third detection line 33 and the quality control line 34.
Comparative example 1
The only difference from example 3 is that: in step S6.1, the treatment solution consists of polyethylene glycol with the mass concentration of 0.5% and water.
Comparative example 2
The only difference from example 3 is that: in step S6.1, the treatment liquid consists of L-phenylalanine with a mass concentration of 0.5% and water.
Comparative example 3
The only difference from example 3 is that: in the step S6.1, the treatment solution is chitosan acidic aqueous solution with the mass concentration of 0.5%, and the pH value of the chitosan acidic aqueous solution is 2-5.
Comparative example 4
The only difference from example 3 is that: in step S6.1, the treatment solution consists of 0.2% lysine, polyethylene glycol with a mass concentration of 0.3% and water.
Comparative example 5
The only difference from example 3 is that: in steps S6.1-S6.2, the nitrocellulose membrane is not modified, and each antibody is directly sprayed on the unmodified nitrocellulose membrane.
Comparative example 6
The only difference from example 3 is that: in step S1, the relevant step of adding sodium borohydride is omitted.
Comparative example 7
The only difference from example 3 is that: in the step (1) of the step S3, a solution was prepared using only 10mg of phthalic dithiol and deionized water without adding lauryl sulfobetaine and benzyl dimethyl hexadecyl ammonium bromide.
Comparative example 8
The only difference from example 3 is that: in the step (1) of the step S2, a solution was prepared using only 50mg of benzyldimethyl hexadecyl ammonium bromide, 10mg of phthalic dithiol and deionized water without adding lauryl sulfobetaine.
The results of the tests of examples 1 to 3 and the respective comparative examples are given below:
table 1 detection time required for detection results in examples and comparative examples
As can be seen from Table 1, the detection time of Candida albicans, aspergillus and Cryptococcus in examples 1-3 was significantly reduced, and the requirement for rapid detection was satisfied, compared with comparative examples, and the detection results of examples 1-3 did not show false positive when the samples contained the species shown in Table 2. Whereas comparative example 6 for the detection of influenza b virus, the first detection line 31 shows false positives in red, no false positives due to the 15nm red gold nanospheres prepared using sodium borohydride in example 3.
Comparative example 7 the black color of the third detection line was lighter when cryptococcus was contained in the sample than in example 3. In comparative example 8, when aspergillus is contained in the sample, the color of the second detection line is light or insufficient to form a blue color visible to the naked eye. In embodiment 3, the color development effect of each detection line is clear and sharp, and the color development effect is stable.
TABLE 2 list of different sample tests
As shown in table 2, other samples were separately tested with the prepared test device, for example: rotavirus, influenza b virus, influenza a virus, staphylococcus aureus, escherichia coli, streptococcus pneumoniae, mycoplasma pneumoniae, and the atopy of each detection line was analyzed by observation results. The detection lines on the reaction pad do not cross react to species other than candida albicans, aspergillus and cryptococcus.
It will be appreciated by persons skilled in the art that the above embodiments are provided for clarity of illustration only and are not intended to limit the scope of the invention. Other variations or modifications will be apparent to persons skilled in the art from the foregoing disclosure, and such variations or modifications are intended to be within the scope of the present invention.
Claims (9)
1. A method of detecting a fungus, wherein the fungus comprises candida albicans, the method comprising:
s1.1, adding gold chloride and trisodium citrate into deionized water, heating and stirring in a magnetic stirring heater to obtain a gold chloride and trisodium citrate mixed solution, wherein the mass ratio of the gold chloride to the trisodium citrate is 4:1; adding sodium borohydride to deionized water until dissolved in an ice bath environment to obtain a sodium borohydride solution of 4 mg/ml; adding sodium borohydride solution into the mixed solution of gold chloride and trisodium citrate, stirring, cooling at room temperature, and standing in a dark environment to turn into red to obtain red color nanosphere solution; wherein the volume of the sodium borohydride solution is 0.25% of the volume of the mixed solution of gold chloride and trisodium citrate;
S1.2, adding candida albicans antibody into pure water and PBS buffer solution, centrifuging and taking supernatant to prepare 6mg/ml candida albicans antibody solution;
S1.3, after the pH value of the red color nanosphere solution is regulated to 9.2, adding the candida albicans antibody solution, mixing and stirring, adding bovine serum albumin with the mass concentration of 10% and polyethylene glycol with the mass concentration of 0.2%, and centrifuging to obtain supernatant; adding a mixed solution of sucrose with the mass concentration of 15% and boric acid with the mass concentration of 0.02% into the supernatant, and obtaining a candida albicans antibody marked red-colored nanosphere solution after volume fixing;
S1.4, spraying the candida albicans antibody marked red color nano ball solution on a glass fiber film to form a bonding pad;
s1.5, respectively diluting candida albicans antibodies and rabbit anti-sheep IgG with PBS buffer solution to a concentration of 3mg/ml, and respectively spraying the candida albicans antibodies and the rabbit anti-sheep IgG on a modified nitrocellulose membrane to form a first detection line and a quality control line on a reaction pad; the preparation of the modified nitrocellulose membrane comprises the following steps: soaking and oscillating a nitrocellulose membrane in a treatment liquid, and then cleaning the nitrocellulose membrane by using distilled water, wherein the treatment liquid consists of 0.2% by mass of L-phenylalanine, 0.3% by mass of polyethylene glycol and water;
S1.6, dripping a sample to be detected to a sample pad, moving the sample to a quality control line and a first detection line of the reaction pad through the combination pad, and determining whether the sample contains candida albicans through color development of the first detection line.
2. The method of detecting according to claim 1, wherein the fungus further comprises aspergillus, the method further comprising:
S2.1, mixing a first mixed aqueous solution of lauryl sulfobetaine and benzyl dimethyl hexadecyl ammonium bromide with a gold salt solution of gold chloride, sequentially adding a silver nitrate solution, an L-ascorbic acid solution and the red gold nanosphere solution obtained in the step S1.1, and standing in a dark environment to turn blue to obtain a blue-colored nanostar solution; wherein the mass ratio of the lauryl sulfobetaine to the benzyl dimethyl hexadecyl ammonium bromide to the gold chloride to the silver nitrate to the L-ascorbic acid is 15:10:10:1:9;
S2.2, adding the aspergillus antibody into pure water and PBS buffer solution, centrifuging and taking supernatant to prepare 6mg/ml aspergillus antibody solution;
S2.3, after the pH value of the blue-colored nanostar solution is adjusted to 9.2, adding the aspergillus antibody solution, mixing and stirring, adding bovine serum albumin with the mass concentration of 10% and polyethylene glycol with the mass concentration of 0.2%, and centrifuging to obtain a supernatant; adding a mixed solution of sucrose with the mass concentration of 15% and boric acid with the mass concentration of 0.02% into the supernatant, and obtaining an aspergillus antibody marked blue-colored nano star solution after volume fixing;
s2.4, spraying the aspergillus antibody marked blue-color nano star solution on the glass fiber film to form the bonding pad;
s2.5, diluting the aspergillus antibody with PBS buffer solution until the concentration is 3mg/ml, spraying the diluted aspergillus antibody on a modified nitrocellulose membrane, and embedding the modified nitrocellulose membrane on a reaction pad to form a second detection line;
S2.6, dripping the sample to be detected to a sample pad, moving the sample to a quality control line and a second detection line of the reaction pad through the combination pad, and determining whether aspergillus is contained in the sample through color development of the second detection line.
3. The method of detection of claim 2, wherein the fungus further comprises cryptococcus, the method of detection further comprising:
S3.1, adding the blue-colored nano-star solution obtained in the step S2.1 into a second mixed aqueous solution of laurylsulfobetaine, benzyl dimethyl hexadecyl ammonium bromide and phthalic dithiol, mixing, adding nano-silver particles, performing ultrasonic dispersion, and standing to obtain a black gold nano-star solution with silver tips; wherein the mass ratio of the lauryl sulfobetaine to the benzyl dimethyl hexadecyl ammonium bromide to the phthalic dithiol to the nano silver particles is 15:10:5:1;
S3.2, adding cryptococcus antibody into pure water and PBS buffer solution, centrifuging, and taking supernatant to prepare 6mg/ml cryptococcus antibody solution;
S3.3, after regulating the pH value of the black gold nanoscin solution with silver at the tip to 9.2, adding the cryptococcus antibody solution, mixing and stirring, adding bovine serum albumin with the mass concentration of 10% and polyethylene glycol with the mass concentration of 0.2%, and centrifuging to obtain supernatant; adding a mixed solution of sucrose with the mass concentration of 15% and boric acid with the mass concentration of 0.02% into the supernatant, and obtaining a gold nanoscin solution with silver at the black tip marked by the cryptococcus antibody after volume fixing;
s3.4, spraying the cryptococcus antibody marked black gold nanoscin solution with silver at the tip onto the glass fiber membrane to form the bonding pad;
s3.5, diluting the cryptococcus antibody with PBS buffer solution until the concentration is 3mg/ml, spraying the cryptococcus antibody on a modified nitrocellulose membrane, and embedding the modified nitrocellulose membrane on a reaction pad to form a third detection line;
S3.6, dripping the sample to be detected to a sample pad, moving the sample to the position of a quality control line and a third detection line of the reaction pad through the combination pad, and determining whether the sample contains cryptococcus or not through color development of the third detection line.
4. The detection method according to claim 1, wherein in step S1.1, the obtained red gold nanosphere solution is stored at a low temperature for later use after being sealed.
5. The method according to claim 3, wherein in step S3.1, the volume ratio of the second mixed aqueous solution to the blue nano-star solution is 25:3.
6. The method according to claim 1, wherein in step S1.3, a potassium carbonate solution with a concentration of 0.3mol/L is added to the red gold nanosphere solution to adjust the pH.
7. The method according to claim 1, wherein in step S1.3, the candida albicans antibody solution is added, mixed and stirred for 30min, bovine serum albumin and polyethylene glycol are added in sequence under stirring, stirring is continued for 40min, and the supernatant is centrifuged.
8. The method according to claim 1, wherein in step S1.4, after forming the conjugate pad, the conjugate pad is vacuum freeze-dried for 24 to 36 hours.
9. A fungus detection device, characterized in that it is applied to the detection method according to any one of claims 1 to 8, comprising a substrate and a sample pad, a binding pad, a reaction pad and a water absorbing pad provided on the substrate, the sample pad having a detection hole for receiving a sample;
One end of the bonding pad is laminated with one end of the sample pad, and the other end of the bonding pad is laminated with one end of the reaction pad;
One end of the water absorption pad is laminated with one end of the reaction pad, which is away from the bonding pad.
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| CN105283761A (en) * | 2013-06-10 | 2016-01-27 | 旭化成纤维株式会社 | Immunochromatographic diagnosis kit |
| CN113607943A (en) * | 2021-08-20 | 2021-11-05 | 浙江嘉孚生物科技有限公司 | Quantum dot fluorescence immunochromatography test paper for simultaneously detecting trichomonas, gardnerella and candida albicans and preparation method thereof |
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