CN111239409B - Up-conversion luminescence immunochromatographic test strip for quantitative detection of O-type foot-and-mouth disease virus antibody and preparation method thereof - Google Patents
Up-conversion luminescence immunochromatographic test strip for quantitative detection of O-type foot-and-mouth disease virus antibody and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an up-conversion luminescence immunochromatographic test strip for quantitative detection of O-type foot-and-mouth disease virus antibodies and a preparation method thereof. The invention uses the Escherichia coli prokaryotic expression technology to express O-type foot-and-mouth disease (O-FMDV) capsid protein, and the O-type foot-and-mouth disease virus-like particles are assembled in vitro to be used as capture antigens; the upconversion luminescence immunochromatographic test strip is prepared by coupling the upconversion luminescence nano material serving as a fluorescent marker with protein G and further combining the fluorescent marker with an antibody by utilizing the special luminescence property of the upconversion luminescence nano material. The immunochromatographic test paper prepared by the invention has high sensitivity, good specificity and repeatability and stable result, can be applied to monitoring the level of the O-type foot-and-mouth disease antibody, and is helpful for further understanding the condition of the O-type foot-and-mouth disease antibody of the hoof-coupled livestock.
Description
Technical Field
The invention relates to an up-conversion luminescence immunochromatographic test strip prepared based on an up-conversion luminescence nano material and a preparation method thereof, and also relates to application of the up-conversion luminescence immunochromatographic test strip in quantitative detection of an O-type foot-and-mouth disease virus antibody. The invention belongs to the field of material chemistry and immunology detection.
Background
The foot and mouth disease virus antigen mainly includes complete virus and its structural protein. The whole virus has the phenomenon of incomplete inactivation, so that the infection risk exists in the use process; and partial structural proteins (such as VP1) have immunogenicity, but have certain structural differences with the complete virus, and the effect of immune response is difficult to guarantee. Virus-like particles (VLPs) are RNA-free empty capsids, have the same structure as whole viruses, have excellent type specificity and immunogenicity, but are not infectious, and are therefore an optimal antigen. There are a number of virus-like particle vaccines available today, such as: hepatitis B virus VLPs vaccines, human papilloma virus VLPs vaccines and hepatitis E virus VLPs vaccines are put into use. Therefore, the development of the foot-and-mouth disease virus-like particle vaccine and the diagnostic reagent thereof has good application prospect.
The colloidal gold immunochromatography technology is characterized in that colloidal gold labeled protein is deposited on a detection line, and qualitative or semi-quantitative analysis is carried out by observing the depth of the deposition line by naked eyes. Due to the limitation of visual observation, the colloidal gold immunochromatographic test strip has poor detection sensitivity and cannot be subjected to quantitative analysis. At present, immunochromatography quantitative analysis methods for luminescent materials or magnetic materials such as quantum dots, rare earth materials, ferroferric oxide and the like are reported.
An upconversion luminescent nanomaterial (UCP) is a special material formed by doping a rare earth element in the crystal lattice, which can convert absorbed low-energy near-infrared photons into high-energy visible light by multiphoton absorption or energy transfer under the excitation of near-infrared light. The up-conversion luminescent material has a different luminescent process from common luminescence, generates fluorescence after being excited by near infrared of 980nm, and has the characteristics of low energy excitation and high energy emission. Under the excitation of 980nm, other biological materials and fluorescent materials can not generate fluorescence emission, and only the up-conversion luminescent material generates fluorescence emission at 520 nm and 580nm, so that the fluorescence signal is detected almost without background interference. The upconversion luminescence has the characteristics of longer fluorescence life, difficult quenching of fluorescence, good biocompatibility, strong tissue penetration capability, low toxicity and the like, so the upconversion luminescence has great potential in the application field of biology.
At present, the most common method for diagnosing foot-and-mouth disease, namely ELISA, has higher sensitivity but longer time consumption, and usually needs more than 1h to collect data. The colloidal gold immunochromatography method has been applied to various fields, but it can perform only qualitative or semi-quantitative detection, and thus, has great limitations. The up-conversion luminescent material replaces colloidal gold as a marker, and the excellent luminescent property of the up-conversion luminescent material is utilized to realize the quantitative detection of the foot-and-mouth disease virus.
Disclosure of Invention
One of the purposes of the invention is to provide a preparation method of an up-conversion luminescence immunochromatographic test strip prepared based on an up-conversion luminescence nano material;
the second purpose of the invention is to establish a method for quantitatively detecting the O-type foot-and-mouth disease virus antibody by using the up-conversion luminescence immunochromatographic test strip.
In order to achieve the purpose, the invention adopts the following technical means:
the invention relates to an up-conversion luminescence immunochromatographic test strip, which comprises a sample pad, a combination pad, an analysis membrane, a water absorption pad and a PVC bottom plate, wherein the sample pad, the combination pad, the analysis membrane and the water absorption pad are sequentially connected, and the PVC bottom plate is positioned below the combination pad and used as an assembly platform;
wherein, the sample pad is used for adding a sample; the binding pad is coated with an up-conversion luminescent nano material coupled with protein G (streptococcal protein G, which is referred to as protein G for short); the analysis membrane is respectively coated with a detection line and a control line sprayed by O-type foot-and-mouth disease virus-like particles and rabbit IgG.
Wherein, preferably, the up-conversion luminescent nano material is NaYF modified by citric acid418% of Yb and 2% of Er nanocrystalline.
Preferably, the citric acid modified upconversion luminescent nanomaterial is prepared by the following steps:
1) synthesis of up-conversion luminescent nano material
a weighing YCl3·6H2O 0.2366g,YbCl3·6H2O0.0775 g and ErCl3·6H2O0.0076 g was added to a 100mL round-bottom flask, and mixed with 6mL Oleic Acid (OA), 15mL Octadecene (ODE); heating to 160 ℃ under the protection of nitrogen to form a uniform solution, and cooling to room temperature; slowSlowly add 10mL of solution containing 0.1g NaOH and 0.148g NH4A methanol solution of F; slowly heating the reaction solution to 50 deg.C for 40min, heating to 100 deg.C for 40min, and removing methanol and water; then, under the protection of nitrogen, heating to 320 ℃ and keeping for 1.5h, after the solution is naturally cooled, adding ethanol for precipitation, washing with ethanol for three times, and dispersing the precipitate in 6mL of cyclohexane to obtain a cyclohexane dispersion liquid containing the nanocrystal for later use;
b in a 100mL round-bottom flask, add NaCOOCF30.135g and Yb (COOCF)3)30.512g, 15mL of Oleic Acid (OA) and 15mL of Octadecene (ODE), dropwise adding the cyclohexane dispersion liquid containing the nanocrystal obtained in the step a, and heating the reaction liquid to 120 ℃ under vacuum for 40 min; then, under the protection of nitrogen, heating to 320 ℃ and keeping for 1.5h, naturally cooling the solution, adding ethanol for precipitation, washing with ethanol for three times, wherein the obtained precipitate is the up-conversion luminescent nano material, and dispersing the precipitate in mL cyclohexane to obtain cyclohexane dispersion of the up-conversion luminescent nano material (UCNP) for later use;
2) surface modification of up-conversion luminescent nanomaterials
0.2g of NOBF was added to a round bottom flask45mL of DMF, 20mg of the above-prepared cyclohexane dispersion of upconversion luminescent material (UCNP) synthesized in step 1) was added thereto, and the mixture was stirred at room temperature for 2 hours. Precipitating with isopropanol and cyclohexane, washing for three times, redispersing the precipitate in DMF, and dropwise adding 1.5mL of 50mg/mL citric acid solution; stirring at room temperature overnight, and centrifuging to obtain citric acid modified up-conversion luminescent nanometer material (UCNP-COOH).
Preferably, the sample pad and the binding pad are made of glass fiber, the analysis membrane is made of nitrocellulose membrane, and the absorbent pad is made of high-density cellulose material.
Preferably, the up-conversion luminescence immunochromatographic test strip is assembled in a plastic shell, a sample adding hole is formed in the shell corresponding to the position of the sample pad, and a visual window is formed in the shell corresponding to the position of the analysis membrane and used for detecting the detection line and the comparison line.
Preferably, the O-type foot-and-mouth disease virus VLPs are assembled from structural proteins VP0, VP1 and VP3 of the O-type foot-and-mouth disease virus.
Furthermore, the invention also provides a method for preparing the up-conversion luminescence immunochromatographic test strip, which comprises the following steps
1) Coupling of up-conversion luminescent nano material and protein G
Adding 200 mu L of upconversion luminescent nano material with the concentration of 10mg/mL, 100 mu L of N-hydroxysuccinimide NHS with the newly prepared concentration of 20mg/mL and 50 mu L of carbodiimide EDC with the newly prepared concentration of 20mg/mL into an EP tube, adding water to 1mL, and carrying out rotary reaction for 1 hour at room temperature; after centrifugal separation, the precipitate is dispersed in water, 160 mu G of protein G is added, and the reaction is carried out for 4 hours at room temperature in a rotating way; centrifuging, dispersing the precipitate, adding bovine serum albumin BSA, and sealing overnight; after centrifugal separation, dispersing in a preservation solution again to obtain the protein G coupled up-conversion luminescent nano material, and refrigerating for later use; the preservation solution is Tris-HCl buffer solution containing 0.5% BSA (w/w) and 0.5% Tween 20 (v/v);
2) scribing a film: diluting the purified O-type foot-and-mouth disease virus VLPs to 0.4mg/mL, diluting rabbit IgG to 1mg/mL, scribing a film on a nitrocellulose membrane by using a special film scribing and gold spraying instrument, taking the VLPs as a detection line, taking the rabbit IgG as a control line, scribing the film with the amount of 0.5 mu L/cm, and placing and airing at room temperature to obtain an analysis membrane for later use;
3) labeling the binding pad: diluting the protein G-coupled up-conversion luminescent nano-material to 2mg/mL, adding 5 mu L of the protein G-coupled up-conversion luminescent nano-material on a bonding pad, and airing at room temperature;
4) assembling the test strip: sequentially sticking a sample pad, a combination pad, an analysis membrane and a water absorption pad on a PVC base plate with an adhesive, overlapping the sample pad, the combination pad, the analysis membrane and the water absorption pad by 1-2mm, assembling the sample pad, the combination pad, the analysis membrane and the water absorption pad into a large card, and cutting the large card into test strips with the width of 3mm by using a slitter;
5) shell installation: the test strip is arranged in a plastic shell, a sample adding hole is arranged on the plastic shell corresponding to the position of the sample pad, a visual window is arranged corresponding to the position of the analysis membrane for detecting the detection line and the comparison line.
Furthermore, the invention also provides application of the up-conversion luminescence immunochromatographic test strip in preparation of a quantitative detection reagent for detecting the O-type foot-and-mouth disease virus antibody.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention uses the up-conversion luminescent nano material as the marker for the first time, and uses the O-type foot-and-mouth disease virus-like particles as the capture antigen, thereby establishing the quantitative detection method of the O-type foot-and-mouth disease;
2. the invention uses the special luminescent property of the up-conversion luminescent nano material, low-energy excitation and high-energy emission, avoids background interference and realizes high-sensitivity detection;
3. the virus-like particles adopted by the invention have a complete structure similar to that of a whole virus, do not carry genetic material RNA, have good type specificity and reactionogenicity, can not be copied, have no infectivity, have no threat of toxin dispersion, and are safe and harmless to operators and the environment;
4. the invention utilizes the characteristics of long fluorescence life and difficult quenching of the up-conversion luminescent nano material, can be stored for a long time, cannot generate photobleaching and has good stability;
5. the immunochromatographic test paper prepared in the invention has high sensitivity, good specificity and repeatability and stable result, can be applied to monitoring of the level of the O-type foot-and-mouth disease antibody, and is helpful for further understanding the condition of the O-type foot-and-mouth disease antibody of the hoof-coupled livestock.
Drawings
FIG. 1 is a Transmission Electron Micrograph (TEM) of an upconversion luminescent nanomaterial;
wherein: (a) up-conversion luminescent nanocrystals (UCNPs), (b) citric acid-modified up-conversion luminescent materials (UCNPs-COOH);
FIG. 2 shows the results of negative and positive sera;
FIG. 3 shows the test results of different concentrations of type-O foot-and-mouth disease antibodies;
FIG. 4 is a linear fit;
the abscissa is the logarithm of the antibody concentration and the ordinate is the logarithm of the signal ratio;
FIG. 5 shows the result of the specificity test.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of Up-conversion luminescence immunochromatographic test strip
1. Preparation and modification of up-conversion luminescent nano material
1) Synthesis of up-conversion luminescent nano material
a NaYF4The synthesis process of 18 percent Yb and 2 percent Er nanocrystals is as follows: weighing YCl3·6H2O 0.2366g,YbCl3·6H2O0.0775 g and ErCl3·6H2O0.0076 g was added to a 100mL round-bottomed flask, and mixed with 6mL Oleic Acid (OA), 15mL Octadecene (ODE). Heating to 160 ℃ under the protection of nitrogen to form a uniform solution, and cooling to room temperature. Slowly add 10mL of solution containing 0.1g NaOH and 0.148g NH4Solution of F in methanol. The reaction solution was slowly heated to 50 ℃ for 40min, then heated to 100 ℃ for 40min, and methanol and water were removed. Then, the mixture was heated to 320 ℃ for 1.5 hours under nitrogen protection. After the solution is naturally cooled, ethanol is added for precipitation, and the solution is washed with ethanol for three times. And dispersing the precipitate in 6mL of cyclohexane to obtain a cyclohexane dispersion liquid containing the nanocrystal for later use.
b in a 100mL round-bottom flask, add NaCOOCF30.135g and Yb (COOCF)3)30.512g, 15mL Oleic Acid (OA) and 15mL Octadecene (ODE). And (c) dropwise adding the cyclohexane dispersion liquid containing the nano crystals obtained in the step (a). The reaction solution was heated to 120 ℃ under vacuum and held for 40 min. Then, the mixture was heated to 320 ℃ for 1.5 hours under nitrogen protection. After the solution is naturally cooled, ethanol is added for precipitation, and the solution is washed with ethanol for three times. The precipitate is the upconversion luminescent nanomaterial (UCNP), and the precipitate is dispersed in 6mL cyclohexane to obtain cyclohexane dispersion of the upconversion luminescent nanomaterial for later use.
2) Surface modification of up-conversion luminescent nanomaterials
0.2g of NOBF was added to a round bottom flask45mL of DMF. 20mg of the cyclohexane dispersion of the upconversion phosphor synthesized in step 1) was added. Stirred at room temperature for 2 h. Precipitation was performed with isopropanol and cyclohexane, and washed three times. The precipitate was redispersed in DMF and 1.5mL of citric acid solution (50mg/mL) was added dropwise. After stirring overnight at room temperature, the mixture was centrifuged for further use. This is the citric acid modified up-conversion luminescent material (UCNP-COOH).
3) Characterization of upconversion luminescent nanomaterials
The prepared up-conversion luminescent nano-material and the up-conversion luminescent nano-material modified by citric acid are characterized by a Transmission Electron Microscope (TEM), a UV-Vis spectrum, a fluorescence spectrum, an infrared spectrum and the like. The TEM is shown in FIG. 1.
Preparation of foot-and-mouth disease Virus-like particles (VLPs) of type O
1) Expression of recombinant VLPs proteins
Competent cells BL21(DE3) -RIL co-transformed by positive recombinant plasmids pSMK/VP0, pSMK/VP1 and pSMA/optiVP3 (the construction method of the positive recombinant plasmid is referred to as CN201610929279.0, the name of the invention is O-type foot-and-mouth disease virus-like particles and the preparation method and the application thereof) stored in the laboratory are inoculated into autoclaved fresh LB liquid culture medium containing 10 mug/mL kanamycin, 50 mug/mL ampicillin and 25 mug/mL chloramphenicol according to the ratio of 1:100, and cultured overnight on a shaker at 37 ℃ and 220 r/min. The overnight cultured expression strain was inoculated into autoclaved fresh LB liquid medium containing 10. mu.g/mL kanamycin, 50. mu.g/mL ampicillin and 25. mu.g/mL chloramphenicol, and shaken at 37 ℃ and 220r/min on a shaker until the OD600 value of the strain liquid became about 0.8. Adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.5mmol/L, inducing at 16 ℃ for 16h, collecting thalli, carrying out ultrasonic disruption, centrifuging and collecting supernatant.
2) Affinity chromatography purification of recombinant VLPs proteins
Collected supernatant and Ni2+The column binding of the affinity chromatography resin is 1 h. First, 10 column volumes of Buffer A wash (20mM Tris-HCl,500mM NaCl,20mM imidazole, 3% Triton X-100, pH 8.0) were usedDeproteinizing, eluting with rinsing solution and Ni2+Column non-specifically bound heteroproteins. Eluting the target protein by using Buffer B washing liquor (20mM Tris-HCl,500mM NaCl,500mM imidazole, 3 per mill TritonX-100, pH 8.0), collecting eluent, and identifying the obtained protein by using an SDS-PAGE electrophoresis experiment. The protein concentration is measured and stored at-80 ℃ for later use.
3) In vitro assembly of VLPs
The fusion protein was cleaved with a small ubiquitination-modified protease, see invitrogen's reagent instructions. Mu.g of the purified fusion protein was digested at 37 ℃ for 30min or overnight at 4 ℃ with 200. mu.L of digestion buffer (50mM Tris-HCl, 150mmol/L NaCl, 0.2% Igepal (NP-40),1mM DTT, pH 8.0), 10. mu.L of small ubiquitin-modified protease (1U/. mu.L). The cleavage mixture was passed through His Trap HP to remove small ubiquitinated modified tag proteins. The label protein cleaved sample was loaded into a dialysis bag with a cut-off molecular weight of 8kD, and the dialysis bag was placed in a 500mL assembly Buffer (250mmol/L NaCl,20 mmol/L Tris-HCl 20mmol/L Imidazole, 1mmol/L CaCl22mmol/L DTT, 0.1% Triton X-100, pH 8.4), the SUMO-tagged structural proteins self-assemble into VLPs.
Collecting the assembled VLPs, concentrating by an ultrafiltration tube with the molecular weight cutoff of 30kDa, and identifying by SDS-PAGE and Western blot, wherein clear bands of VP0, VP1 and VP3 can be seen; the VLPs prepared by observing after staining with 3% phosphotungstic acid by a Transmission Electron Microscope (TEM) are spherical, the size is 20-30nm, and the obtained protein is determined to be the VLPs of the O-type foot-and-mouth disease virus.
3. Coupling of up-conversion luminescent nano material and protein G
In an EP tube, 200. mu.L of the upconverting luminescent nanomaterial prepared in step 1 (10mg/mL), 100. mu.L of freshly prepared N-hydroxysuccinimide NHS (20mg/mL) and 50. mu.L of freshly prepared carbodiimide EDC (20mg/mL) were added, water was added to 1mL, and the reaction was rotated at room temperature for 1 hour. After centrifugal separation, the precipitate is dispersed in water, 160 mu G of protein G is added, and the reaction is carried out for 4 hours at room temperature in a rotating way; after centrifugation and redispersion of the pellet, blocking with bovine serum albumin BSA was carried out overnight. After centrifugal separation, the mixture is redispersed in a preservation solution (Tris-HCl buffer solution containing 0.5 percent BSA (w/w) and 0.5 percent Tween 20 (v/v)) to obtain the up-conversion luminescent nano material coupled with the protein G, and the up-conversion luminescent nano material is refrigerated and preserved for later use.
4. Assembly of up-conversion luminescence immunochromatographic test strip
1) Structure of up-conversion luminescence immunochromatographic test strip
The immunochromatographic test strip comprises a PVC base plate, a sample pad, a combination pad, an analysis membrane and a water absorption pad. Glass fibers are sequentially stuck on the PVC base plate with the adhesive to serve as a sample pad for adding samples; the glass fiber is used as a combination pad and is used for adding the up-conversion luminescent nano material of the coupled protein G; the nitrocellulose membrane is used as an analysis membrane, and O-type foot-and-mouth disease virus VLPs and rabbit IgG are coated on the nitrocellulose membrane and respectively used as a detection line and a control line; the high-density cellulose is used as a water absorption pad, and the sample liquid continuously flows on the test strip through capillary action. The test strip is assembled in the plastic shell, a sample adding hole is formed in the shell corresponding to the position of the sample pad, a visual window is arranged corresponding to the position of the analysis membrane, the detection line and the comparison line can be observed, and signals can be tested through an instrument.
2) And (6) scribing the film. The prepared and purified O-type foot-and-mouth disease virus VLPs are diluted to 0.4mg/mL, and the rabbit IgG is diluted to 1 mg/mL. And scratching a membrane on the nitrocellulose membrane by using a special membrane scratching gold spraying instrument, wherein VLPs are used as detection lines, rabbit IgG is used as a control line, the scratching amount is 0.5 mu L/cm, and the analysis membrane is placed at room temperature and dried for later use.
3) And (6) assembling the test strip. And sequentially sticking the sample pad, the bonding pad, the analysis membrane and the water absorption pad on the PVC base plate with the adhesive, and overlapping 1-2mm to assemble the large card. The test strips were then cut to a width of 3mm with a slitter.
4) The conjugate pad is labeled. The up-conversion luminescent nanomaterial coupled with the protein G is diluted to 2 mg/mL. 5 mu L of up-conversion luminescence nano-material coupled with protein G is added on the bonding pad of the test strip and dried at room temperature.
5) And (5) casing. The test strip is enclosed in a plastic shell.
6) The using method comprises the following steps: if the blood sample contains O-type foot-and-mouth disease antibodies, the serum is diluted properly and then added on a sample pad of a test strip, liquid reaches a combination pad along the test strip through the capillary action of a water absorption pad and a microporous membrane, the O-type foot-and-mouth disease antibodies contained in the sample are combined with protein G marked by an up-conversion luminescent material on the combination pad to form a compound, the compound is continuously moved to an analysis membrane and is captured by the O-type foot-and-mouth disease VLPs at a detection line, and meanwhile, the up-conversion luminescent material is deposited on the detection line to generate a fluorescent signal; protein G which is not bound to the antibody continues to move forward, and binds to rabbit IgG at the control line, and the upconverting material is deposited on the control line, generating a fluorescent signal. If the detection line and the control line do not generate fluorescence signals, the test strip is indicated to be invalid. The results of the negative and positive tests are shown in FIG. 2.
Example 2 evaluation of application of Up-conversion luminescence immunochromatographic test strip
Immune chromatography quantitative method for O type foot-and-mouth disease virus antibody
The O-type foot-and-mouth disease antibody is diluted by the diluent to different concentrations of 0.1, 0.3, 1, 3, 30 and 50 mu g/mL. And respectively adding the antibodies with different concentrations into the sample adding holes of the up-conversion luminescence immunochromatography test strip, detecting on a tester after 20min, and recording the result. The concentrations and signal ratios were plotted logarithmically, respectively, to establish a linear standard curve. Each batch of immunochromatographic test paper needs to correspond to a standard curve. The test results of different concentrations of the type-O foot-and-mouth disease antibody are shown in FIG. 3.
2. Sensitivity of the probe
And respectively taking logarithms of the concentration and the signal value of the O-type foot-and-mouth disease antibody, then drawing, performing linear fitting, and calculating the Cutoff value of 10 blank tests to obtain the detection limit of the up-conversion luminescence immunochromatographic test strip of 0.34 mug/mL, as shown in figure 4.
3. Specificity of
The antiserum of O-type foot-and-mouth disease virus, A-type foot-and-mouth disease virus, porcine circovirus type 2, seneca virus, porcine pseudorabies virus, porcine encephalitis B virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine parvovirus is respectively taken to be tested by the up-conversion luminescence immunochromatographic test strip, the result is shown in figure 5, except that the signal value of the O-type foot-and-mouth disease virus is high, the signal values of other antiserum are very low, and the up-conversion luminescence immunochromatographic test strip is proved to have good specificity on the O-type foot-and-mouth disease.
4. Stability of
The prepared up-conversion luminescence immunochromatographic test strip was stored at 37 ℃ and tested at different time intervals. In addition, 3 different concentrations of the foot-and-mouth disease virus type O antibody were tested according to the use method established in example 1, each concentration being set to 3-5 replicates (5 replicates within a batch, 3 replicates between batches). The result shows that the up-conversion luminescence immunochromatographic test strip has good repeatability and stable result.
Claims (6)
1. The up-conversion luminescence immunochromatographic test strip is characterized by comprising a sample pad, a combination pad, an analysis membrane, a water absorption pad and a PVC (polyvinyl chloride) bottom plate which is positioned below the sample pad, the combination pad, the analysis membrane and the water absorption pad which are sequentially connected;
wherein, the sample pad is used for adding a sample; the binding pad is coated with an up-conversion luminescent nano material coupled with protein G; the analysis membrane is respectively coated with a detection line and a control line sprayed by O-type foot-and-mouth disease virus-like particles and rabbit IgG;
the up-conversion luminescent nano material is NaYF modified by citric acid418% of Yb and 2% of Er nanocrystalline;
the citric acid modified up-conversion luminescent nano material is prepared by the following steps:
1) synthesis of up-conversion luminescent nano material
a weighing YCl3·6H2O 0.2366g,YbCl3·6H2O0.0775 g and ErCl3·6H2O0.0076 g was added to a 100mL round-bottom flask, and mixed with 6mL Oleic Acid (OA), 15mL Octadecene (ODE); heating to 160 ℃ under the protection of nitrogen to form a uniform solution, and cooling to room temperature; slowly add 10mL of solution containing 0.1g NaOH and 0.148g NH4A methanol solution of F; slowly heating the reaction solution to 50 deg.C for 40min, heating to 100 deg.C for 40min, and removing methanol and water; then, under the protection of nitrogen, heating to 320 ℃ and keeping for 1.5h, after the solution is naturally cooled, adding ethanol for precipitation, washing with ethanol for three times, and dispersing the precipitate in 6mL of cyclohexane to obtain a cyclohexane dispersion liquid containing the nanocrystal for later use;
b in a 100mL round-bottom flask, add NaCOOCF30.135g and Yb (COOCF)3)30.512g, 15mL of Oleic Acid (OA) and 15mL of Octadecene (ODE), dropwise adding the cyclohexane dispersion liquid containing the nanocrystal obtained in the step a, and heating the reaction liquid to 120 ℃ under vacuum for 40 min; then, under the protection of nitrogen, heating to 320 ℃ and keeping for 1.5h, naturally cooling the solution, adding ethanol for precipitation, washing with ethanol for three times, wherein the obtained precipitate is the up-conversion luminescent nano material, and dispersing the precipitate in 6mL of cyclohexane to obtain cyclohexane dispersion liquid of the up-conversion luminescent nano material for later use;
2) surface modification of up-conversion luminescent nanomaterials
0.2g of NOBF was added to a round bottom flask45mL of DMF, adding the cyclohexane dispersion liquid of the up-conversion luminescent material of 20mg synthesized in the step 1), and stirring for 2 hours at room temperature; and precipitating with isopropanol and cyclohexane, washing for three times, re-dispersing the precipitate in DMF, dropwise adding 1.5mL of 50mg/mL citric acid solution, stirring at room temperature overnight, and performing centrifugal separation to obtain the citric acid modified up-conversion luminescent nano material.
2. The strip of claim 1, wherein the sample pad and the conjugate pad are made of glass fiber, the analytical membrane is made of nitrocellulose, and the absorbent pad is made of high density cellulose material.
3. The strip of claim 1, wherein the strip is mounted in a plastic housing, and the housing has wells corresponding to the sample pads and windows corresponding to the analytical membrane for viewing the detection and control lines.
4. The upconversion luminescence immunochromatographic test strip according to claim 1, wherein the O-type foot-and-mouth disease virus-like particle is assembled from structural proteins VP0, VP1 and VP3 of the O-type foot-and-mouth disease virus.
5. A method for preparing the up-conversion luminescence immunochromatographic test strip of any one of claims 1 to 4, comprising the following steps
1) Synthesis of up-conversion luminescent nano material
a weighing YCl3·6H2O 0.2366g,YbCl3·6H2O0.0775 g and ErCl3·6H2O0.0076 g was added to a 100mL round-bottom flask, and mixed with 6mL Oleic Acid (OA), 15mL Octadecene (ODE); heating to 160 ℃ under the protection of nitrogen to form a uniform solution, and cooling to room temperature; slowly add 10mL of solution containing 0.1g NaOH and 0.148g NH4A methanol solution of F; slowly heating the reaction solution to 50 deg.C for 40min, heating to 100 deg.C for 40min, and removing methanol and water; then, under the protection of nitrogen, heating to 320 ℃ and keeping for 1.5h, after the solution is naturally cooled, adding ethanol for precipitation, washing with ethanol for three times, and dispersing the precipitate in 6mL of cyclohexane to obtain a cyclohexane dispersion liquid containing the nanocrystal for later use;
b in a 100mL round-bottom flask, add NaCOOCF30.135g and Yb (COOCF)3)30.512g, 15mL of Oleic Acid (OA) and 15mL of Octadecene (ODE), dropwise adding the cyclohexane dispersion liquid containing the nanocrystal obtained in the step a, and heating the reaction liquid to 120 ℃ under vacuum for 40 min; then, under the protection of nitrogen, heating to 320 ℃ and keeping for 1.5h, naturally cooling the solution, adding ethanol for precipitation, washing with ethanol for three times, wherein the obtained precipitate is the up-conversion luminescent nano material, and dispersing the precipitate in 6mL of cyclohexane to obtain cyclohexane dispersion liquid of the up-conversion luminescent nano material for later use;
2) surface modification of up-conversion luminescent nanomaterials
0.2g of NOBF was added to a round bottom flask45mL of DMF, adding the cyclohexane dispersion liquid of the up-conversion luminescent material of 20mg synthesized in the step 1), and stirring for 2 hours at room temperature; precipitating with isopropanol and cyclohexane, washing for three times, dispersing the precipitate in DMF again, dropwise adding 1.5mL of 50mg/mL citric acid solution, stirring at room temperature overnight, and performing centrifugal separation to obtain the citric acid modified up-conversion luminescent nano material;
3) coupling of up-conversion luminescent nano material and protein G
Adding 200 mu L of the upconversion luminescent nano material prepared in the step 2) with the concentration of 10mg/mL into an EP tube, adding 100 mu L of newly prepared N-hydroxysuccinimide NHS with the concentration of 20mg/mL and 50 mu L of newly prepared carbodiimide EDC with the concentration of 20mg/mL, adding water to 1mL, and carrying out rotary reaction for 1 hour at room temperature; after centrifugal separation, the precipitate is dispersed in water, 160 mu G of protein G is added, and the reaction is carried out for 4 hours at room temperature in a rotating way; centrifuging, dispersing the precipitate, adding bovine serum albumin BSA, and sealing overnight; after centrifugal separation, dispersing in a preservation solution again to obtain the protein G coupled up-conversion luminescent nano material, and refrigerating for later use; the preservation solution is Tris-HCl buffer solution containing 0.5% BSA (w/w) and 0.5% Tween 20 (v/v);
4) scribing a film: diluting the purified O-type foot-and-mouth disease virus VLPs to 0.4mg/mL, diluting rabbit IgG to 1mg/mL, scribing a membrane on a nitrocellulose membrane by using a special membrane scribing and gold spraying instrument, taking O-type foot-and-mouth disease virus-like particles as a detection line, taking the rabbit IgG as a control line, scribing the membrane at the amount of 0.5 mu L/cm, and placing and airing at room temperature to obtain an analysis membrane for later use;
5) labeling the binding pad: diluting the protein G-coupled up-conversion luminescent nano-material to 2mg/mL, adding 5 mu L of the protein G-coupled up-conversion luminescent nano-material on a bonding pad, and airing at room temperature;
6) assembling the test strip: sequentially sticking a sample pad, a combination pad, an analysis membrane and a water absorption pad on a PVC base plate with an adhesive, overlapping the sample pad, the combination pad, the analysis membrane and the water absorption pad by 1-2mm, assembling the sample pad, the combination pad, the analysis membrane and the water absorption pad into a large card, and cutting the large card into test strips with the width of 3mm by using a slitter;
7) shell installation: the test strip is arranged in a plastic shell, a sample adding hole is arranged on the plastic shell corresponding to the position of the sample pad, a visual window is arranged corresponding to the position of the analysis membrane for detecting the detection line and the comparison line.
6. The use of the up-conversion luminescence immunochromatographic test strip of any one of claims 1 to 4 in the preparation of a quantitative detection reagent for detecting an O-type foot-and-mouth disease virus antibody.
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