CN115216288B - High-sensitivity double-flux detection immunochromatography test strip based on double-excitation orthogonal emission up-conversion luminescence nano-particles - Google Patents

High-sensitivity double-flux detection immunochromatography test strip based on double-excitation orthogonal emission up-conversion luminescence nano-particles Download PDF

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CN115216288B
CN115216288B CN202210582109.5A CN202210582109A CN115216288B CN 115216288 B CN115216288 B CN 115216288B CN 202210582109 A CN202210582109 A CN 202210582109A CN 115216288 B CN115216288 B CN 115216288B
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CN115216288A (en
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刘芳芳
李春霞
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Weifang University of Science and Technology
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Abstract

The invention provides a high-sensitivity double-flux detection immunochromatography test strip based on double-excitation orthogonal emission up-conversion luminescent nano particles. The up-conversion luminescence nano-particles can emit red light or green light at any one wave band under the excitation of two different near infrared lights at the same time, namely, one near infrared excitation light can excite light of one wave band. The sample pad of the double-flux immunochromatographic test strip is arranged in the center of the bottom plate, and the two sides of the sample pad are symmetrically and sequentially adhered with the binding pad, the nitrocellulose membrane and the absorbent paper. Unlike one-way test paper, the double-pass test paper has gold grains coupled with ochratoxin A and vomitoxin antibody fixed separately to the two end combining pads and up-converting nanometer luminous grains coupled with ochratoxin A and vomitoxin antigen fixed onto corresponding T line. The invention can realize high-sensitivity and rapid detection of two detection substances. The method has the advantages of simple operation, low cost, low detection background value, stable signal and higher sensitivity.

Description

High-sensitivity double-flux detection immunochromatography test strip based on double-excitation orthogonal emission up-conversion luminescence nano-particles
Technical Field
The invention belongs to the technical field of biomedical diagnosis, and particularly relates to a high-sensitivity double-flux detection immunochromatography test strip based on double-excitation orthogonal emission up-conversion luminescence nano particles.
Background
Food safety is becoming a global issue with the growth of intensive agriculture and the food industry; the health reliability of food reaching the consumer's hand should be determined in a quick, economical, accurate manner, especially in terms of contaminant screening. In order to meet the requirements of rapid and on-site analysis, the development of biosensors for food quality control is receiving increasing attention.
Ochratoxin A and vomitoxin have become the main factors contaminating cereal and byproducts in China, and have synergistic effects with various toxins. They are widely found in nature, wherein wheat and corn are the most main contaminated crops, and even if the wheat and corn are ingested by people and livestock, the wheat and corn have strong nephrotoxicity, mutagenicity, teratogenicity and carcinogenicity, and the toxins reduce the immunity proliferation of lymphoid organs, reduce the whole immunity function, influence the humoral immunity response and generate negative feedback effect on cellular immunity, and the targeted detoxification method has proved to be unable to remove the pollution and harm of the two toxins effectively due to the high stability of the two toxins, so the basic research force must be increased to develop a more effective detection method to eliminate the damage of the mycotoxins from the source.
The immunoassay method plays a crucial role in the current rapid mycotoxin screening due to the simplicity, easiness and high specificity. Compared with an ELISA method, the immunoassay method based on the colloidal gold nanoparticles (AuNP) is simple and quick in sample preparation method, can obtain a result within 3-10 min, does not need complicated sample pretreatment, trained personnel and expensive and precise instruments, and is more suitable for on-site quick detection.
Upconverting nanoparticles are inorganic nanocrystals that convert near infrared light into shorter wavelength emissions. This unique photophysical property, coupled with the narrow emission bandwidth and large anti-stokes shift, makes UCNP an excellent optical label for biosensing, as they can be detected without optical background interference of the sample matrix, receiving increasing attention in improving detection performance.
Therefore, UCNPs are combined with traditional colloidal gold immunochromatography, a double-throughput immunochromatography test strip based on a UCNP-Au composite system is developed through the arrangement of a bidirectional T line, the simultaneous detection of ochracin A and vomitoxin is realized, the cost can be greatly saved, and the convenience and the sensitivity are improved.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a high-sensitivity double-flux detection immunochromatography test strip based on double-excitation orthogonal emission up-conversion luminescent nano particles. The up-conversion luminescence nano particles can emit red light or green light at any one wave band under the excitation of two different near infrared lights, namely, one near infrared excitation light can excite light of one wave band, the light can be combined with colloidal gold, and a brand-new double-flux detection mode is created by utilizing the bidirectional diffusion characteristic of liquid drops so as to realize multiple substance detection in food. The invention can realize single-line detection of various detection substances, and has simple and convenient operation, low cost and higher sensitivity.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
the preparation method of the dual-excitation orthogonal emission up-conversion luminescent nanoparticle is characterized by comprising the following steps of:
(1) Preparing a double-excitation orthogonal emission up-conversion luminescent nanoparticle with a three-layer core-shell structure by adopting a seed crystal method, wherein NaYF 4 nanoparticle doped with Yb, tm and Er is taken as an inner core, a first shell layer of Yb-doped NaYF 4 is wrapped outside the inner core, and a second shell layer of Yb-doped NaYF 4 and Nd-doped NaYF 4:Yb/Tm/Er@NaYF4:Yb@NaYF4 which is co-doped with Yb, tm, er and Nd is wrapped outside the first shell layer, so that a Yb/Nd core-shell structure is formed; the water-soluble dual-excitation orthogonal emission up-conversion luminescent nano-particles UCNPs are prepared, and the up-conversion luminescent nano-particles emit red light under the excitation of 980nm near infrared light and emit green light under the excitation of 808nm near infrared light.
The preparation method of the dual-excitation orthogonal emission up-conversion luminescent nanoparticle UCNPs is characterized by further comprising the following steps:
(2) Preparation of UCNPs-antigen: the antigen of the detection object is marked on the surface of the water-soluble dual-excitation orthogonal emission up-conversion luminescent nanoparticle through physical adsorption and the like, and the dual-excitation orthogonal emission up-conversion luminescent nanoparticle marked with the antigen of the detection object is obtained.
The preparation method of the gold nanoparticle is characterized by comprising the following steps of:
(1) Preparing a colloidal gold solution:
0.25mL of 0.1M chloroauric acid solution was added to 100mL of ultrapure water, and heated under magnetic stirring to reflux to boiling. 1.5mL of 1% sodium citrate solution was added rapidly and reflux continued for about 30 minutes. During which the color of the solution changed from pale yellow to black and then to deep red. And (3) cooling to room temperature, and filtering the prepared wine-red gold nanoparticle sol by using a filter membrane with the diameter of 0.22 mu m to obtain gold nanoparticles.
The preparation method of the gold nanoparticle AuNPs is characterized by further comprising the following steps:
(2) Preparation of AuNPs-antibodies: the gold nanoparticle labeled with the antibody of the object to be detected is obtained by labeling the antibody of the object to be detected on the surface of the gold nanoparticle through physical adsorption and the like.
The dual-excitation orthogonal emission up-conversion luminescent nanoparticle prepared by the method is characterized in that the up-conversion luminescent nanoparticle can emit any one of red light or green light wave bands under the excitation of two different near infrared lights at the same time, namely, one near infrared excitation light can excite light of one wave band.
The high-sensitivity double-flux detection immunochromatography test strip based on the double-excitation orthogonal emission up-conversion luminescent nano particles is characterized in that a sample pad is arranged in the center of a bottom plate, and two sides of the sample pad are sequentially adhered with a binding pad, an NC film and water absorbing paper. The assembly sequence is as follows: firstly, arranging two detection tracks on a nitrocellulose membrane, scribing a treated UCNPs solution onto an NC membrane along a specific track to form two test lines, symmetrically assembling the NC membrane on a bottom plate, and then sequentially adhering a bonding pad, water absorbing paper and a sample pad, wherein the sample pad is assembled at the center of a test strip; gold nanoparticles are arranged on the binding pad, and antibodies of the objects to be detected are marked on the gold nanoparticles; an antigen of a detection object marked by the dual-excitation orthogonal emission up-conversion luminescence nano particle is immobilized on the nitrocellulose membrane.
The high-sensitivity double-flux detection immunochromatographic test strip is characterized in that the up-conversion luminescent nano particles can emit any one of red light or green light under the excitation of two different near infrared lights, and only two test lines for fixing a detection object marked by the double-excitation orthogonal emission up-conversion luminescent nano particles are required to be marked on a nitrocellulose membrane of the chromatographic test strip, so that signals of one wave band of the double-excitation orthogonal emission up-conversion nano particles under the near infrared excitation light are used as detection T lines, and signals of the other wave band can be used as quality control C lines, thereby achieving the effect of multiple bidirectional single-line detection.
The high-sensitivity double-flux detection immunochromatography test strip is characterized in that a sample pad of the double-flux test strip is arranged in the center of a bottom plate, a binding pad, an NC film and absorbent paper are adhered to the two sides of the sample pad in a symmetrical sequence, the high-sensitivity double-flux detection immunochromatography test strip is different from a unidirectional detection test strip, gold particles coupled with ochratoxin A and vomitoxin antibody are respectively fixed on the binding pads at the two ends of the double-flux test strip, and UCNPs coupled with the ochratoxin A and vomitoxin antigen are fixed on corresponding T lines.
The high-sensitivity double-flux detection immunochromatography test strip for the double-excitation orthogonal emission up-conversion luminescence nano-particles is characterized in that: the commercial nitrocellulose membrane was 60cm in size; the bonding pad is made of two glass fibers with the length of 5mm, the length of the sample pad is 10mm, and the sample pad is stuck to the middle position of the nitrocellulose membrane during assembly.
The detection method of the colloidal gold immunochromatographic test strip for fluorescence quenching of the double-excitation double-emission up-conversion luminescence nano-particles is characterized by comprising the following steps of:
(1) When the prepared chromatographic test strip does not drop the sample to be detected on the binding pad, the two different detection signals are obtained by exciting the antigen of the detection object marked by the luminescent nanoparticle through the double-excitation orthogonal emission up-conversion fixed on the nitrocellulose membrane through 980nm and 808nm lasers respectively.
(2) When the sample does not contain detection substances, the sample is dripped on the binding pad, and all the gold nanoparticles flow into the nitrocellulose membrane and are combined with the antigen of the detection substances marked by the double-excitation orthogonal emission up-conversion luminescence nanoparticles fixed on the membrane. Because the absorption area (520 nm) of the gold nanoparticles is exactly coincident with the green light emission area (540 nm) of the up-conversion luminescence nanoparticles under 808nm laser excitation, the gold nanoparticles can quench the green light of the up-conversion luminescence nanoparticles; however, the red light emission area (660 nm) of the up-conversion luminescence nano-particles is not affected, so that a red light signal under 980nm laser excitation can be used as a quality control C line of a detection sample;
The step (2) further comprises the following steps:
During detection, solutions of detection objects with different concentrations are respectively dripped on the sample pad for detection. The detection sample can be combined with the antibody corresponding to the object to be detected marked on the gold nano-particles, the detection sample can form an aggregate with a part of the gold nano-particles, the aggregate is left on the combination pad, and the other part of the gold nano-particles can flow into the nitrocellulose membrane along with the rest detection sample to two sides and are combined with the antigen of the object to be detected marked by the double-excitation orthogonal emission up-conversion luminescent nano-particles fixed on the nitrocellulose membrane. Because only a part of the gold nanoparticles are combined with the antigen of the detection object marked by the dual-excitation orthogonal emission up-conversion luminescence nanoparticles fixed on the nitrocellulose membrane, the gold nanoparticles cannot completely quench the green light of the up-conversion luminescence nanoparticles, and the quenching effect gradually decreases along with the increase of the concentration of the detection object, namely the green light signal gradually increases. At this time, the green light signal under 808nm laser excitation can be used as a detection T line for detecting the sample. The fluorescence intensity of the T line and the C line of the quality inspection are obtained through 980nm and 808nm laser excitation, the ratio of the fluorescence intensity of the T line to the fluorescence intensity of the C line is established, and the quantitative detection of the two components in the sample to be inspected is realized through the comparison with a standard curve.
The beneficial effects of the invention are mainly as follows:
1. The double-flux detection immunochromatography test strip provided by the invention only comprises two detection lines, has a simple structure, is a novel immunochromatography test strip capable of rapidly detecting two mycotoxins, mainly utilizes double-excitation orthogonal emission up-conversion luminescence nano particles to have two signals, and can realize single-line detection of two detection substances after being combined with colloidal gold; test strip stability: can be stably maintained for at least 3 years.
2. The preparation method of the up-conversion luminescent nano-particles based on the dual-excitation orthogonal emission has the advantages of simple process and easy implementation; the dual-excitation dual-emission up-conversion luminescent nanoparticle has 980nm excitation red light and 808nm excitation green light; the particle size is small: d is approximately equal to 30nm; the dispersity is good: PDI <0.3.
3. According to the detection method of the double-flux detection immunochromatography test strip, UCNPs are used as a biological marker labeled antibody, the luminous results of two T lines can be directly observed under the excitation of near infrared light, and the background interference is eliminated due to the unique optical characteristics of the UCNPs, the background value is low, the signal is stable, and the sensitivity is greatly improved; and UCNPs in the test paper are harmless to testers and the environment, and have good safety.
4. According to the detection method of the double-flux detection immunochromatography test strip, more pretreatment is not needed for a sample to be detected, quantitative measurement can be directly realized by means of the up-conversion luminescence sensor, the operation is simple and quick, the on-site operation can be realized, the detection is quick within 3-10 min, and the result accuracy is high.
Drawings
FIG. 1 is a transmission electron microscope image of a dual excitation orthogonal emission up-conversion nanoparticle;
FIG. 2 is a schematic reaction diagram of an immune reaction;
FIG. 3 is a schematic diagram of the detection principle of the high-sensitivity dual-throughput detection immunochromatography test strip based on dual-excitation orthogonal emission up-conversion luminescence nano-particles.
FIG. 4 is a schematic diagram of the detection result of a high-sensitivity dual-throughput detection immunochromatography test strip based on dual-excitation orthogonal emission up-conversion luminescent nano-particles.
FIG. 5 is a schematic diagram of a high-sensitivity dual-throughput detection immunochromatographic test strip based on dual-excitation orthogonal emission up-conversion luminescent nanoparticles.
Detailed Description
Referring to fig. 1 to 5, the following detailed description of the technical solution of the present invention is given by way of examples and drawings.
Example 1:
The preparation method of the dual-excitation orthogonal emission up-conversion luminescent nanoparticle provided by the embodiment is used for preparing the dual-excitation orthogonal emission up-conversion luminescent nanoparticle with 980nm excitation red light and 808nm excitation green light, then combining with colloidal gold to prepare a dual-throughput detection immunochromatography test strip, and simultaneously detecting ochratoxin A and vomitoxin, and comprises the following steps:
1. Preparation of UCNPs
1.1 Preparing a double-excitation orthogonal emission up-conversion luminescent nanoparticle with a three-layer core-shell structure by adopting a seed crystal method, wherein NaYF 4 nanoparticle doped with Yb, tm and Er is taken as an inner core, a first shell layer of Yb doped NaYF 4 is wrapped outside the inner core, and a second shell layer of Yb and Nd doped NaYF 4 is wrapped outside the first shell layer to form a Yb, tm, er and Nd co-doped NaYF 4:Yb/Tm/Er@NaYF4:Yb@NaYF4 of a Yb/Nd core-shell structure; the water-soluble dual-excitation orthogonal emission up-conversion luminescent nano-particles UCNPs are prepared, and the up-conversion luminescent nano-particles emit red light under the excitation of 980nm near infrared light and emit green light under the excitation of 808nm near infrared light.
The step (1) specifically comprises the following steps:
a. Synthesizing NaYF 4 nano-particles doped with Yb, tm and Er as inner cores
10ML of Oleic Acid (OA), 10mL of Octadecene (ODE), 0.84g of NaF solid and 3 0.0683g、Tm(OAc)3 0.0017g、Er(OAc)3 0.2752g of Yb (OAc) are weighed and added into a three-neck flask A, the temperature is raised to 110-120 ℃ by magnetic stirring, the temperature is kept for 10min, and then the vacuum is pumped to remove water and oxygen; after the removal, introducing N 2, heating to 300 ℃, and reacting for 1h;
b. Wrapping a first shell layer of Yb doped NaYF 4 outside the inner core
4ML of Oleic Acid (OA), 4mL of Octadecene (ODE), 0.4788g of NaF solid and 3 0.07.07 g of Yb (OAc) are weighed and added into a three-neck flask B, the temperature is raised to 110-120 ℃ by magnetic stirring, the mixture is kept for 10min, and then the water and oxygen are removed by vacuumizing; introducing N 2 after the removal, heating to 150 ℃, and after the reaction in the step a is finished, injecting the mixture into the three-neck flask A at the speed of 0.13mL/min by using a needle tube, and reacting for 1h at 300 ℃;
c. a second shell layer wrapping Yb and Nd outside the first shell layer
Weighing 4mL of Oleic Acid (OA), 4mL of Octadecene (ODE) and 3 0.0525g、Nd(OAc)3 0.4333g of Yb (OAc), adding into a three-neck flask C, magnetically stirring and heating to 110-120 ℃, keeping for 10min, and vacuumizing to remove water and oxygen; introducing N 2 after the removal, and after the reaction in the step b is finished, injecting the mixture into the three-neck flask A at the speed of 0.13mL/min by using a needle tube, and reacting for 100min at 300 ℃; cooling to room temperature after the reaction is finished, and centrifuging the reaction liquid in the three-neck flask A in a centrifuge tube to separate out the obtained nano particles;
d. Stripping oleic acid from the nanoparticle surface obtained in step c
Taking the nano particles obtained in the step c, adding a mixed solution of ethanol and concentrated hydrochloric acid (7.5 mL of ethanol and 62.5 mu L of concentrated hydrochloric acid) with pH=1 into the nano particles, carrying out ultrasonic dispersion uniformly, vibrating while carrying out ultrasonic treatment for 30min, centrifuging, removing supernatant, adding a mixed solution of ethanol and concentrated hydrochloric acid (7.5 mL of ethanol and 7.5mL of concentrated hydrochloric acid) with pH=4 into the obtained nano particles, carrying out ultrasonic dispersion uniformly, vibrating while carrying out ultrasonic treatment for 30min, carrying out centrifugal separation again, and washing the obtained nano particles to obtain the water-soluble double-excitation orthogonal emission up-conversion luminescent nano particles UCNPs.
2. Preparation of UCNPs-antigen
The pH of the mixed solution is adjusted to about 8.5 by adding 0.02moL/L of K 2CO3 solution into the water-soluble dual-excitation orthogonal emission up-conversion luminescent nano particles with the concentration of 1 mg/mL. 10. Mu.g of AFB1 antigen was added to the mixed solution, and after mixing homogeneously for 30 seconds, incubation was performed in a shaker at 37℃for 23 minutes with shaking. 200. Mu.L of BSA (mass fraction: 10%) solution and 200. Mu.L of PEG 20000 (mass fraction: 1%) solution were then added to the mixed solution and incubation was continued in a shaker at 110℃for 15min with shaking. And (3) placing the mixed solution into a centrifuge tube, centrifuging for 30min at the temperature of 13000r/min and the temperature of 4 ℃, and dispersing the mixed solution into a PBS buffer solution containing 1% BSA and having the concentration of 0.01M, pH =7.0, thereby completing the preparation of the solution of the double-excitation orthogonal emission up-conversion luminescent nano particles marked with the antigen of the AFB1 to be detected.
3. Preparing a colloidal gold solution:
0.25mL of 0.1M chloroauric acid solution was added to 100mL of ultrapure water, and heated under magnetic stirring to reflux to boiling. 1.5mL of 1% sodium citrate solution was added rapidly and reflux continued for about 30 minutes. During which the color of the solution changed from pale yellow to black and then to deep red. And (3) cooling to room temperature, and filtering the prepared wine-red gold nanoparticle sol by using a filter membrane with the diameter of 0.22 mu m to obtain gold nanoparticles.
4. Preparation of AuNPs-antibodies
10ML of the prepared gold nanoparticles were collected and concentrated to 2mL by centrifugation. 0.02moL/L K 2CO3 solution was added to 1mg/mL gold nanoparticles, and the pH of the mixed solution was adjusted to about 8.5. 10. Mu.g of AFB1 antibody was added to the mixed solution, and after mixing homogeneously for 30 seconds, incubation was performed in a shaker at 110℃for 20 minutes with shaking. 200. Mu.L of BSA (10% by mass) and 200. Mu.L of PEG 20000 (1% by mass) were added to the mixture and incubation was continued with shaking in a shaker at 37℃for 15min. The mixed solution is placed in a centrifuge tube, centrifuged for 30min at 13000r/min and 4 ℃, and then dispersed in PBS buffer solution containing 1% BSA and 0.01M, pH =7.0, thus completing the preparation of the gold nanoparticle solution of the antibody of AFB1 to be detected.
5. The preparation of the chromatographic test strip has a specific structure shown in figure 5.
Two pieces of round glass fibers (conjugate pads) having a width of 5mm were immersed in an antibody-Au NP solution having a concentration of 4mg/mL for 10 minutes, and then dried at 37℃for 2 hours. Antigen (OTA-BSA, DON-BSA) conjugated UCNPs solution was diluted to 4mg/mL and streaked onto NC membrane along a specific distance at a concentration of 1.5. Mu.L/cm, respectively, to form two symmetrical T lines, which were further dried at 37℃for 2 hours. The sample pad is assembled in the center of the test strip. Finally, the assembled test strips were cut to size and stored in a refrigerator at 4 ℃ for future use.
The commercial nitrocellulose membrane was 60cm in size; the combination pad is a circular glass fiber with the width of 5mm, the diameter of the sample pad is 10mm, and the sample pad is stuck to the middle position of the test strip during assembly.
The high-sensitivity double-flux detection immunochromatography test strip based on the double-excitation orthogonal emission up-conversion luminescence nano-particles further comprises the following steps:
(1) Treatment of bond pads
Taking a glass cellulose film as a bonding pad material, firstly soaking the bonding pad in a bonding pad treatment liquid for 24 hours, drying at 37 ℃, putting the bonding pad into the solution of the double gold nanoparticles marked with the OTA and DON antibodies to be detected obtained in the step 1, soaking for 10 minutes, and drying at 37 ℃ to finish the treatment of the bonding pad; the conjugate pad treatment was a PBS buffer solution containing 1% bas, 2% tween-20 and 5% sucrose at a mass concentration of 0.01M, pH =7.4.
(2) Treatment of nitrocellulose membranes
Marking a specific annular orbit of a solution of the double-excitation orthogonal emission up-conversion luminescence nano particles marked with the antigen of OTA and DON to be detected on a nitrocellulose membrane with a parameter of 1.5 mu L/cm, and drying to finish the treatment of the nitrocellulose membrane;
(3) Assembly of test strips
And (3) adhering the treated bonding pad to the center position of each test ring on the nitrocellulose membrane, thus completing the assembly of the multiple double-throughput detection test strip.
6. Detection method and process of colloidal gold immune test strip
(1) When the prepared chromatographic test strip does not drop the sample to be detected on the sample pad, the two different detection signals are obtained by exciting the antigens of the detection object marked by the luminescent nano particles through the double-excitation orthogonal emission up-conversion fixed on the nitrocellulose membrane through 980nm and 808nm lasers respectively.
(2) When the sample does not contain detection substances, the sample is dripped on a sample pad, and all gold nanoparticles flow into nitrocellulose membranes towards two sides and are combined with antigens of the detection substances marked by the double-excitation orthogonal emission up-conversion luminescence nanoparticles fixed on the nitrocellulose membranes. Because the absorption area (520 nm) of the gold nanoparticles is exactly coincident with the green light emission area (540 nm) of the up-conversion luminescence nanoparticles under 808nm laser excitation, the gold nanoparticles can quench the green light of the up-conversion luminescence nanoparticles; however, the red light emission area (660 nm) of the up-conversion luminescence nano-particles is not affected, so that a red light signal under 980nm laser excitation can be used as a quality control C line of a detection sample;
The step (2) further comprises the following steps:
During detection, solutions of detection objects with different concentrations are respectively dripped on the sample pad for detection. The detection sample can be combined with the antibody corresponding to the object to be detected marked on the gold nano-particles, the detection sample can form an aggregate with a part of the gold nano-particles, the aggregate is left on the combination pad, and the other part of the gold nano-particles and the rest of the detection sample flow into the nitrocellulose membrane together and are combined with the antigen of the object to be detected marked by the double-excitation orthogonal emission up-conversion luminescent nano-particles fixed on the membrane. Because only a part of the gold nanoparticles are combined with the antigen of the detection object marked by the dual-excitation orthogonal emission up-conversion luminescence nanoparticles fixed on the nitrocellulose membrane, the gold nanoparticles cannot completely quench the green light of the up-conversion luminescence nanoparticles, and the quenching effect gradually decreases along with the increase of the concentration of the detection object, namely the green light signal gradually increases. At this time, the green light signal under 808nm laser excitation can be used as a detection T line for detecting the sample. The fluorescence intensity of the T line and the C line of the quality inspection are obtained through 980nm and 808nm laser excitation, the ratio of the fluorescence intensity of the T line to the fluorescence intensity of the C line are established, and the quantitative detection of two components (OTA and DON) contained in the sample to be detected is realized through the comparison with a standard curve.
The foregoing description of the exemplary embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any modifications, equivalents, and alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims (9)

1. A high-sensitivity double-flux detection immunochromatography test strip is characterized in that the test strip is prepared by adopting double-excitation orthogonal emission up-conversion luminescent nano particles; the sample pad of the high-sensitivity double-throughput detection immunochromatography test strip is arranged in the center of a bottom plate, two sides of the sample pad are symmetrically and sequentially adhered with a binding pad, a nitrocellulose membrane and absorbent paper, and unlike a unidirectional detection test strip, gold nanoparticles coupled with ochratoxin A and vomitoxin antibodies are respectively fixed on the binding pads at two ends of the high-sensitivity double-throughput detection immunochromatography test strip, and double-excitation orthogonal emission up-conversion nanoparticles coupled with the ochratoxin A and vomitoxin antigens are fixed on corresponding T lines, so that when a sample to be detected is dripped, the solution to be detected migrates to two sides at the same speed; the dual-excitation orthogonal emission up-conversion luminescent nano particles can emit red light or green light in any one of two wave bands under the excitation of two different near infrared lights, namely, light in one wave band can be excited by one near infrared excitation light energy;
the dual-excitation orthogonal emission up-conversion luminescent nano-particles are prepared by the following steps:
(1) Preparing a double-excitation orthogonal emission up-conversion luminescent nanoparticle with a three-layer core-shell structure by adopting a seed crystal method, wherein NaYF 4 nanoparticle doped with Yb, tm and Er is taken as an inner core, a first shell layer of Yb-doped NaYF 4 is wrapped outside the inner core, and a second shell layer of Yb-doped NaYF 4 and Nd-doped NaYF 4:Yb/Tm/Er@NaYF4:Yb@NaYF4 which is co-doped with Yb, tm, er and Nd is wrapped outside the first shell layer, so that a Yb/Nd core-shell structure is formed; the water-soluble dual-excitation orthogonal emission up-conversion luminescent nano-particles UCNPs are prepared, and the up-conversion luminescent nano-particles emit red light under the excitation of 980nm near infrared light and emit green light under the excitation of 808nm near infrared light.
2. The high-sensitivity double-flux detection immunochromatographic test strip according to claim 1, which is characterized in that the up-conversion luminescent nano-particles can emit red light or green light under the excitation of near infrared light of two different wave bands at the same time, and only two antigen detection lines T lines of detection objects marked by the double-excitation orthogonal emission up-conversion luminescent nano-particles are required to be marked on a nitrocellulose film of the chromatographic test strip, so that signals of one wave band emitted by the double-excitation orthogonal emission up-conversion luminescent nano-particles under the near infrared excitation light are taken as detection T lines, and signals of the other wave band are taken as quality control C lines, thereby achieving the effect of bidirectional single-line detection.
3. The high-sensitivity double-throughput detection immunochromatographic test strip according to claim 1, wherein a sample pad of the high-sensitivity double-throughput detection immunochromatographic test strip is arranged in the center of a bottom plate, a binding pad, an NC film and absorbent paper are adhered in a bilateral symmetry sequence, and unlike a unidirectional detection test strip, gold particles coupled with ochratoxin A and vomitoxin antibodies are respectively fixed on the binding pads at the two ends of the double-throughput test strip, and UCNPs coupled with ochratoxin A and vomitoxin antigens are fixed on corresponding T lines.
4. The high-sensitivity dual-throughput detection immunochromatographic test strip according to claim 1, wherein: the size of the nitrocellulose membrane is 60 cm; the bonding pad is made of two pieces of glass fibers with the length of 5mm, the length of the sample pad is 10mm, and the sample pad is stuck to the middle position of the nitrocellulose membrane during assembly.
5. The high-sensitivity dual-throughput detection immunochromatographic strip of claim 1, wherein the preparation of the dual-excitation orthogonal emission up-conversion luminescent nanoparticle UCNPs further comprises the steps of:
(2) Preparation of UCNPs-antigen: the antigen of the detection object AFB1 is marked on the surface of the water-soluble dual-excitation orthogonal emission up-conversion luminescent nanoparticle through physical adsorption, so that the dual-excitation orthogonal emission up-conversion luminescent nanoparticle marked with the antigen of the detection object AFB1 is obtained.
6. The high-sensitivity double-throughput detection immunochromatographic test strip according to claim 1, wherein the gold nanoparticles are prepared by the following steps:
(1) Preparing a colloidal gold solution:
0.25 Adding the solution of the chloroauric acid with the concentration of 0.1M into the ultrapure water with the concentration of 100mL M, heating and refluxing to boiling under magnetic stirring, rapidly adding the solution of the sodium citrate with the concentration of 1.5mL and continuously refluxing for 30 minutes; the color of the solution changes from light yellow to black and then to dark red; and (3) cooling to room temperature, and filtering the prepared wine-red gold nanoparticle sol by using a filter membrane with the diameter of 0.22 mu m to obtain gold nanoparticles.
7. The high-sensitivity dual-throughput detection immunochromatographic test strip of claim 6, wherein the preparation of gold nanoparticle AuNPs further comprises the following steps:
(2) Preparation of AuNPs-antibodies: and labeling the antibody of the detection object AFB1 on the surface of the gold nanoparticle through physical adsorption to obtain the gold nanoparticle labeled with the antibody of the detection object AFB 1.
8. A detection method using the high-sensitivity dual-throughput detection immunochromatographic test strip according to any one of claims 1 to 7, comprising the steps of:
(A) When the prepared immunochromatographic test strip does not drop the sample to be detected in the center of the binding pad, the two different detection signals are obtained by exciting antigens of the detection object marked by the luminescent nanoparticle through the double-excitation orthogonal emission up-conversion on the nitrocellulose membrane through 980nm and 808nm lasers respectively;
(B) When the sample does not contain detection substances, the sample is dripped in the center of the sample pad, and the gold nanoparticles flow into two sides of the nitrocellulose membrane along with the sample and are combined with the antigen of the detection substances marked by the double-excitation orthogonal emission up-conversion luminescence nanoparticles fixed on the membrane; because the absorption area of the gold nanoparticles is exactly coincident with the green light emission area of the up-conversion luminescence nanoparticles under 808nm laser excitation, the gold nanoparticles can quench the green light of the up-conversion luminescence nanoparticles; however, the red light emission area of the up-conversion luminescence nano-particles is not affected, so that the red light signal excited by 980nm laser is used as a quality control C line of the detection sample.
9. The method of claim 8, wherein the step (B) further comprises the steps of:
During detection, solutions of detection objects with different concentrations are respectively dripped on a sample pad for detection, a detection sample is combined with antibodies corresponding to detection objects marked on gold nanoparticles on two sides, the detection sample forms an aggregate with a part of gold nanoparticles and is left on the combination pad, and the other part of gold nanoparticles flows into a nitrocellulose membrane together with the rest detection sample and is combined with antigens of the detection objects marked by the double-excitation orthogonal emission up-conversion luminescence nanoparticles fixed on the membrane; because only a part of the gold nanoparticles are combined with the antigen of the detection object marked by the dual-excitation orthogonal emission up-conversion luminescence nanoparticles fixed on the nitrocellulose membrane, the gold nanoparticles cannot completely quench the green light of the up-conversion luminescence nanoparticles, and the quenching effect gradually decreases along with the increase of the concentration of the detection object, namely the green light signal gradually increases; at this time, the green light signal under 808nm laser excitation is used as a detection T line for detecting the sample; the fluorescence intensity of the T line and the C line of the quality inspection are obtained through 980nm and 808nm laser excitation, the ratio of the fluorescence intensity of the T line to the fluorescence intensity of the C line is established, and the quantitative detection of the two components in the sample to be inspected is realized through the comparison with a standard curve.
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