CN111505279B - Method for silver growth enhanced ultrasensitive detection of rotavirus - Google Patents
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Abstract
The invention relates to the technical field of immunochromatography analysis, in particular to a method for detecting rotavirus by silver growth enhancement, which introduces a controllable silver growth technology to amplify the optical signal intensity of a test strip detection line colloidal gold probe on the basis of a traditional colloidal gold immunochromatography test strip, and comprises the following steps: (1) preparing a colloidal gold immunochromatography test strip; (2) preparing silver growth solution; and (3) amplifying the colorimetric signal for controlling silver growth. The invention has the advantages of simple operation, high sensitivity, good stability, high detection speed and readable results, does not need other auxiliary instruments and equipment, and can be used for rapidly and ultra-sensitively detecting rotavirus on site in clinic and emergencies and large-scale epidemiological investigation.
Description
Technical Field
The invention relates to the technical field of immunochromatography analysis, in particular to a method for detecting rotavirus by silver growth enhancement ultrasensitive.
Background
Colloidal gold immunochromatography test strips are widely applied to the fields of clinical diagnosis, food safety detection, environmental monitoring and the like due to simplicity, rapidness and convenience and naked eye detection. The traditional colloidal gold immunochromatography test strip mainly adopts colloidal gold with the particle size of 20-40 nm as a signal probe, however, the colorimetric signal intensity is relatively weak due to the smaller particle size of the colloidal gold immunochromatography test strip, and the detection sensitivity is low. The relatively low detection sensitivity limits to some extent its use in ultrasensitive detection. In recent years, with the rapid development of nanoscience and technology, various novel nano materials such as fluorescent microspheres, quantum dots, quantum dot microspheres, up-conversion fluorescent nanoparticles, carbon nano materials and magnetic materials have been reported to be used as detection probes instead of colloidal gold to improve the detection sensitivity of traditional immunochromatographic test strips, but the synthesis of these nano probes is relatively complex, expensive and difficult to popularize and use by markets. Compared with the novel nano materials, the colloidal gold has the advantages of simple synthesis, easy modification, good biocompatibility, stable optical property, easy reading and the like, and occupies a main position in the immunochromatography test strip, particularly in commercial production, and the market share of the colloidal gold reaches over 90 percent. Therefore, how to improve the detection sensitivity of the traditional colloidal gold immunochromatography test strip has important significance for further expanding the application of the colloidal gold immunochromatography test strip in ultrasensitive detection.
The detection line signal amplification strategy based on the test strip does not need to synthesize additional auxiliary signal amplification materials, so that the scientific research workers are widely concerned. The strategy mainly comprises the following categories: 1) Double gold signal amplification technique: on the basis of the traditional colloidal gold immunochromatography test strip, a signal amplification gold label pad is introduced, so that the gold label pad can specifically detect the conjugate of the alloy label antibody by means of antigen-antibody reaction, biotin-streptavidin system and the like, and further aggregation of colloidal gold nano particles is caused, and a colorimetric signal of the colloidal gold is amplified, such as patent CN102507929A; 2) Enzyme enhancement technique: on the basis of the traditional colloidal gold immunochromatography test strip, horseradish peroxidase or nano enzyme with peroxidase activity is introduced to catalyze and oxidize corresponding substrates such as tetramethyl benzidine (TMB) and the like to generate colored precipitate which is deposited on a detection line, so that colorimetric signals of colloidal gold are amplified; 3) Gold or silver staining enhancement techniques: on the basis of the traditional colloidal gold immunochromatography test strip, gold or silver ions are easy to take colloidal gold as a core when a reducing agent exists, and are aggregated on the surface of the colloidal gold to be reduced into gold or silver atoms, so that the colloidal gold with nanoscale size is grown into larger particles through aggregation and reduction of gold or silver ions, and the colorimetric signals of the colloidal gold are amplified, as in patent CN102135536A. The former two methods often easily affect the immunity of the colloidal gold probe due to the need of double labeling the colloidal gold probe, resulting in poor methodological stability. The growth of the metal nano shell layer in the gold or silver staining enhancement technology belongs to a layer-by-layer growth mode, the growth rate of the metal nano shell layer is closely related to the number of gold nano particles on the T, C line of the test strip, and the concentration of the growth liquid and the growth time, so that the growth process is uncontrollable, the stability and the reproducibility of the detection result are poor, and the popularization and the application of the method in practice are greatly limited. Furthermore, these methods have limited ability to increase detection sensitivity, on the order of only 1 to 2 orders of magnitude. Therefore, the sensitized colloidal gold immunochromatographic test strip still cannot realize the ultrasensitive detection of the low-concentration to-be-detected object. The invention provides a method for remarkably enhancing the optical signal intensity of a colloidal gold probe on a test strip detection line by using a polymer-mediated controllable silver growth technology, the color of a strip is changed from none to existence and from weak to strong, and naked eye qualitative detection under the condition of ultralow target concentration can be realized. No public literature report on the sensitivity of the detection of the traditional colloidal gold immunochromatographic test strip sensitized by adopting the controllable silver growth technology is found through the search.
Disclosure of Invention
Aiming at the current defect of low detection sensitivity caused by weak signal intensity of a 20-40 nm colloidal gold probe of the traditional colloidal gold immunochromatographic test strip, the invention uses polymer-mediated colloidal gold probe surface controllable silver growth deposition to obviously amplify the optical signal intensity of the colloidal gold probe on a test strip detection line, provides a novel technology for improving the detection sensitivity of the traditional colloidal gold immunochromatographic method, and realizes the on-site rapid detection of an ultralow-concentration target detection object.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the invention provides a method for detecting rotavirus by silver growth enhancement ultrasensitive, which comprises the following steps:
(1) Preparing a colloidal gold immunochromatography test strip, and detecting rotavirus by adopting a double-antibody sandwich method;
(2) Preparing a silver growth solution: a) Silver growth solution A: accurately weighing 200-400 mg of silver nitrate and 100-200 mg of polymer, respectively dissolving in 10mL of ultrapure water, and then mixing the two to prepare silver growth A solution; b) Silver growth solution B: accurately weighing 1-2 g of vitamin C sodium solution and 1mg of trisodium citrate, respectively dissolving in 10mL of ultrapure water and 1mL of ultrapure water, and then mixing the two to prepare silver growth solution B;
(3) And (3) amplifying a colorimetric signal for controllable silver growth: a) Accurately taking 100-120 mu L of sample solution to be detected by a liquid transfer device, adding the sample solution into a sample adding hole of a detection card, and reacting for 5-10 min at room temperature; b) Washing the reacted test strip sequentially by using phosphate buffer solution and ultrapure water, and soaking the test strip in silver growth A solution for reaction for 5 minutes at room temperature; c) And transferring the test strip to silver growth solution B for continuous reaction for 2min, and carrying out macroscopic observation of an experimental result to carry out rapid qualitative analysis.
Further, the preparation of the colloidal gold immunochromatographic test strip in the step (1) comprises the following steps:
1) Preparation of colloidal gold-labeled rotavirus detection antibody: 1mL of citric acid coated colloidal gold solution with the particle size of 30-40 nm is taken, and the pH value of the solution is regulated to 7.0-8.0 by using potassium carbonate solution with the concentration of 0.2M; adding 5 mu L rotavirus detection antibody solution with the concentration of 1-2 mg/mL into the solution, and carrying out oscillation reaction for 30min at room temperature; then adding 100 mu L of 1-2 mg/mL bovine serum albumin solution into the mixed solution, and continuing to perform oscillation reaction for 30min at room temperature; centrifuging at 10000rpm for 20min at 4deg.C, discarding supernatant, and redissolving the precipitate in 0.01M Phosphate Buffer Solution (PBS) with pH of 7.4 to obtain final product colloidal gold labeled rotavirus detection antibody;
2) Preparing a colloidal gold immunochromatography test strip: a) The preparation of the bonding pad is to spray the rotavirus detection antibody marked by colloidal gold on the glass fiber film; b) The preparation of the nitrocellulose membrane with a detection line and a quality control line comprises the steps of respectively spraying a rotavirus capture antibody solution with the concentration of 1-2 mg/mL and a donkey anti-mouse secondary antibody solution with the concentration of 1-2 mg/mL on the nitrocellulose membrane to draw two parallel lines, wherein the streak of the rotavirus capture antibody solution is used as the detection line (T line), and the streak of the donkey anti-mouse secondary antibody solution is used as the quality control line (C line); c) Assembling a colloidal gold immunochromatography test strip: and (3) respectively and sequentially adhering the sample pad, the binding pad with the gold-labeled detection antibody, the nitrocellulose membrane with T and C lines and the absorbent paper to a plastic lining plate, cutting the adjacent overlapping parts with the length of 2mm into test strips with the width of 4mm, sealing, drying and preserving at the temperature.
Further, in the step (2), the polymer is polyethylenimine PEI, polyacrylic acid PAA, polyvinylpyrrolidone PVP or polylysine PL.
The invention also provides a kit for enhancing the ultrasensitive detection of rotavirus by utilizing silver growth, which comprises a colloidal gold immunochromatography test strip and silver growth solution.
The invention relates to a polymer-mediated controllable silver growth signal amplification technology, which relates to controllable reduction deposition of sodium ascorbate induced bivalent silver ions under the mediation of a polymer on the surface of a colloidal gold probe. The principle is that a colloidal gold test strip after conventional detection is soaked in silver growth solution A and silver growth solution B in sequence, and at the moment, pre-adsorbed silver ions are reduced into silver simple substances to be deposited on the surface of a colloidal gold probe, so that signal enhancement is realized. In the scheme, the quantity of the silver nano particles which are reduced and deposited in the detection area is related to the quantity of the pre-adsorbed silver ions, and the quantity of the pre-adsorbed silver ions is only related to the quantity of the colloidal gold probes in the detection area of the test strip, so that the controllable growth of the silver nano particles can be realized. Compared with the traditional gold and silver growth sensitization type test strip, the method has higher sensitivity and better accuracy and reliability of the detection result. In addition, the silver nitrate, the polymer, the vitamin C sodium solution and the trisodium citrate which are involved in the signal amplification system are all commercially produced on a large scale, so that the method has the advantages of low reagent cost, high stability and good reproducibility, and has a good commercial prospect.
The invention is suitable for ultrasensitive detection of disease-related antigens and antibody markers, wherein the disease-related markers comprise: tumor markers such as carcinoembryonic antigen, prostate specific antigen, alpha fetoprotein; markers of inflammation such as C-reactive protein, procalcitonin, interleukin 6; myocardial markers such as troponin, myoglobin, N-terminal atrial natriuretic peptide, and N-terminal brain natriuretic peptide; infectious disease markers such as novel coronavirus antigen and antibody, HIV antigen and antibody, ebola virus antigen and antibody, middle east respiratory syndrome coronavirus antigen and antibody, hepatitis B virus-related antigen and antibody; pathogens such as viruses, pathogenic bacteria and the like, and is particularly suitable for on-site rapid ultrasensitive detection of objects to be detected. Sample treatment is carried out according to a conventional treatment method.
The technical scheme of the invention has the following beneficial effects:
1. the invention adopts polymer-mediated controllable silver growth signal amplification technology based on the traditional colloidal gold immunochromatography test strip to amplify the colorimetric signal intensity of the colloidal gold probe combined on the detection line, thereby improving the sensitivity of detecting rotavirus of the colloidal gold immunochromatography test strip and retaining the characteristic of strong specificity.
2. The sensitization immunochromatography test strip constructed by the invention is particularly suitable for ultrasensitive detection of trace target analytes, and has wide application prospects in clinical and emergency field detection, such as early screening of infectious diseases and the like.
Drawings
FIG. 1 (A) is a structural diagram of a traditional colloidal gold immunochromatographic test strip, and (B) is a schematic diagram of the method of the present invention;
FIG. 2 is a standard curve of rotavirus quantitative detection in a labeled sample before and after sensitization in example 1 of the present invention.
Detailed Description
Hereinafter, embodiments of the present invention will be described in detail. In order to avoid unnecessary detail, well-known structures or functions will not be described in detail in the following embodiments.
Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The test reagent consumables used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified.
In the following experiments, phosphate buffer (PBS, 005m, ph 7.4) was configured as follows: naCl 40g, na 2 HPO 4 13.5g，KH 2 PO 4 1.0g of KCl 1.0g was dissolved in 1L of ultrapure water. The pH was adjusted with 0.1M NaOH.
The rotavirus detection antibody and the capture antibody involved in the assay and the donkey anti-mouse secondary antibody were supplied by Abcam corporation. The silver nitrate, polyethylenimine PEI, sodium ascorbate solution and trisodium citrate are all commercially available.
Example 1
A detection kit for a sensitization type colloidal gold immunochromatography test strip comprises the colloidal gold immunochromatography test strip and a colorimetric signal amplification reagent; as shown in a fig. 1 a, the colloidal gold immunochromatographic test strip comprises a sample pad, a binding pad, an NC membrane and absorbent paper, wherein the binding pad is provided with a rotavirus detection antibody marked by colloidal gold, and the nitrocellulose membrane is provided with a detection line (T line) and a quality control line (C line); the colorimetric signal amplification reagent comprises silver growth solution A and silver growth solution B.
The kit is prepared by the following steps:
1. preparation of colloidal gold immunochromatography test strip
1. Preparation of colloidal gold-labeled rotavirus detection antibody:
1mL of citric acid coated colloidal gold solution with the particle size of 35nm is taken, and the pH value of the solution is regulated to 7.4 by potassium carbonate solution with the concentration of 0.2M; adding 5 mu L rotavirus detection antibody solution with the concentration of 1mg/mL into the solution, and carrying out oscillation reaction for 30min at room temperature; then adding 100 mu L of 1mg/mL bovine serum albumin solution into the mixed solution, and continuing to perform oscillation reaction for 30min at room temperature; centrifuging at 10000rpm for 20min at 4deg.C, discarding supernatant, and redissolving the precipitate in 0.01M Phosphate Buffer Solution (PBS) with pH of 7.4 to obtain final product colloidal gold labeled rotavirus detection antibody.
2. Preparing a colloidal gold immunochromatography test strip:
a) The preparation of the bonding pad is to spray the rotavirus detection antibody marked by colloidal gold on the glass fiber film;
b) The preparation of the nitrocellulose membrane with a detection line and a quality control line comprises the steps of respectively spraying a rotavirus capture antibody solution with the concentration of 1mg/mL and a donkey anti-mouse secondary antibody solution with the concentration of 1mg/mL on the nitrocellulose membrane to draw two parallel lines, wherein the rotavirus capture antibody solution is marked as a detection line (T line), and the donkey anti-mouse secondary antibody solution is marked as a quality control line (C line);
c) Assembling a colloidal gold immunochromatography test strip: and (3) respectively and sequentially adhering the sample pad, the binding pad with the gold-labeled detection antibody, the nitrocellulose membrane with T and C lines and the absorbent paper to a plastic lining plate, cutting the adjacent overlapping parts with the length of 2mm into test strips with the width of 4mm, sealing, drying and preserving at the temperature.
2. Preparation of silver growth solution
a) Silver growth solution A: accurately weighing 300mg of silver nitrate and 150mg of polyethyleneimine PEI, respectively dissolving in 10mL of ultrapure water, and then mixing the two to prepare silver growth A solution;
b) Silver growth solution B: accurately weighing 1.5g of vitamin C sodium solution and 1mg of trisodium citrate, respectively dissolving in 10mL of ultrapure water and 1mL of ultrapure water, and then mixing the two to prepare silver growth solution B.
3. Colorimetric signal amplification treatment for controllable silver growth
a) Accurately taking 100 mu L of standard sample solution containing rotaviruses with different concentrations (1152000, 576000, 288000, 57600, 11520, 2304, 460, 92, 18 PFU/mL) by using a pipette, adding the standard sample solution into a sample adding hole of a test strip detection card, and reacting for 5min at room temperature;
b) Washing the reacted test strip sequentially by using phosphate buffer solution and ultrapure water, and soaking the test strip in silver growth A solution for reaction for 3 minutes at room temperature;
c) And transferring the test strip to silver growth solution B for continuous reaction for 2min, judging the experimental result by naked eyes, and reading the optical density values of the T and C lines in a colloidal gold reader.
Comparative example 1
Using a traditional test strip, 100 mu L of standard sample solution containing rotaviruses with different concentrations (1152000, 576000, 288000, 57600, 11520, 2304, 460, 92 and 18 PFU/mL) is accurately taken by a liquid-transfering device, added into a sample adding hole of a test strip detection card for reaction, the experimental result is judged by naked eyes without silver growth liquid treatment, and the optical density values of T and C lines are read by a colloidal gold reader.
As shown in FIG. 2, the conventional test strip has a detection sensitivity of 11520PFU/mL and a regression equation of y= 55.893ln (x) -412.7 (R 2 =0.990), y represents the T-line optical density value, x represents the virus concentration; the linear detection range is 11520-1152000PFU/mL; the detection sensitivity of the sensitized colloidal gold test strip reaches 18PFU/mL, and the regression equation is y= 61.802ln (x) -52.102 (R) 2 =0.9930), y represents the T-line optical density value, x represents the virus concentration; the linear detection range is 18-288000PFU/mL. The result shows that the sensitization test strip provided by the invention has higher detection sensitivity and a quicker and wider linear detection range, and is suitable for ultra-sensitive detection of rotavirus with extremely low concentration.
Conclusion: the detection test described in example 2 shows that the novel method provided by the invention is more suitable for ultrasensitive detection of trace rotavirus in a sample, and further proves that the novel method based on the polymer-mediated controllable silver growth technology for sensitizing the traditional colloidal gold immunochromatographic test strip is feasible.
Although the invention uses rotavirus as a model analyte to discuss the feasibility of enhancing the detection sensitivity of the immunochromatographic test strip by using the polymer-mediated controllable silver growth technology on a double-antigen sandwich immunochromatographic platform, the limitation of the invention is not limited to rotavirus, and other target analytes such as biological macromolecules, nucleic acid, pathogens and the like detected by using the method of the invention are all within the protection scope of the patent. In addition, in addition to using conventional antigen-antibody reactions, the present invention can also be used in the detection of target analytes by constructing corresponding ultrasensitive analytical methods using novel biological recognition reactions such as nucleic acid hybridization, nucleic acid aptamers, ligand receptors, etc., which have appeared in recent years. Therefore, the development and extension of the present invention based on these recognition molecules are also within the scope of the present invention.
Claims (3)
1. A method for silver growth enhanced ultrasensitive detection of rotavirus, comprising the steps of:
(1) Preparing a colloidal gold immunochromatography test strip, and detecting rotavirus by adopting a double-antibody sandwich method;
(2) Preparing a silver growth solution: a) Silver growth solution A: accurately weighing 200-400 mg of silver nitrate and 100-200 mg of polyethyleneimine, respectively dissolving in 10mL of ultrapure water, and then mixing the two to prepare silver growth A solution; b) Silver growth solution B: accurately weighing 1-2 g of vitamin C sodium solution and 1mg of trisodium citrate, respectively dissolving in 10mL of ultrapure water and 1mL of ultrapure water, and then mixing the two to prepare silver growth solution B;
(3) And (3) amplifying a colorimetric signal for controllable silver growth: a) Accurately taking 100-120 mu L of sample solution to be detected by a liquid transfer device, adding the sample solution into a sample adding hole of a detection card, and reacting for 5-10 min at room temperature; b) Washing the reacted test strip sequentially by using phosphate buffer solution and ultrapure water, and soaking the test strip in silver growth A solution for reaction for 5 minutes at room temperature; c) And transferring the test strip to silver growth solution B for continuous reaction for 2min, and carrying out macroscopic observation of an experimental result to carry out rapid qualitative analysis.
2. The method for ultra-sensitively detecting rotavirus by silver growth enhancement according to claim 1, wherein the preparation of the colloidal gold immunochromatographic test strip in the step (1) comprises the following steps:
1) Preparation of colloidal gold-labeled rotavirus detection antibody: 1mL of citric acid coated colloidal gold solution with the particle size of 30-40 nm is taken, and the pH value of the solution is regulated to 7.0-8.0 by using potassium carbonate solution with the concentration of 0.2M; adding 5 mu L rotavirus detection antibody solution with the concentration of 1-2 mg/mL into the solution, and carrying out oscillation reaction for 30min at room temperature; then adding 100 mu L of 1-2 mg/mL bovine serum albumin solution into the mixed solution, and continuing to perform oscillation reaction for 30min at room temperature; centrifuging at 10000rpm for 20min at 4deg.C, discarding supernatant, and redissolving the precipitate in 0.01M phosphate buffer solution PBS (phosphate buffer solution) with pH of 7.4 to obtain colloidal gold labeled rotavirus detection antibody;
2) Preparing a colloidal gold immunochromatography test strip: a) The preparation of the bonding pad is to spray the rotavirus detection antibody marked by colloidal gold on the glass fiber film; b) The preparation of the nitrocellulose membrane with a detection line and a quality control line comprises the steps of respectively spraying a rotavirus capture antibody solution with the concentration of 1-2 mg/mL and a donkey anti-mouse secondary antibody solution with the concentration of 1-2 mg/mL on the nitrocellulose membrane to draw two parallel lines, wherein a rotavirus capture antibody solution streak is used as a detection line T line, and a donkey anti-mouse secondary antibody solution streak is used as a quality control line C line; c) Assembling a colloidal gold immunochromatography test strip: and (3) respectively and sequentially adhering the sample pad, the binding pad with the gold-labeled detection antibody, the nitrocellulose membrane with T and C lines and the absorbent paper to a plastic lining plate, cutting the adjacent overlapping parts with the length of 2mm into test strips with the width of 4mm, sealing, drying and preserving at the temperature.
3. A kit for a method for ultrasensitive detection of rotavirus using silver growth enhancement as claimed in claim 1, characterized in that the kit comprises a colloidal gold immunochromatographic test strip and a silver growth solution.
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