CN115216288A - High-sensitivity double-flux detection immunochromatographic test strip based on double-excitation orthogonal emission up-conversion luminescent nanoparticles - Google Patents

High-sensitivity double-flux detection immunochromatographic test strip based on double-excitation orthogonal emission up-conversion luminescent nanoparticles Download PDF

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CN115216288A
CN115216288A CN202210582109.5A CN202210582109A CN115216288A CN 115216288 A CN115216288 A CN 115216288A CN 202210582109 A CN202210582109 A CN 202210582109A CN 115216288 A CN115216288 A CN 115216288A
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CN115216288B (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 immunochromatographic test strip based on double-excitation orthogonal emission up-conversion luminescent nanoparticles. The up-conversion luminescent nano-particles can simultaneously emit red light or green light in any one waveband under the excitation of two different near-infrared lights, namely, one near-infrared excitation light can excite light in one waveband. A sample pad of the double-flux immunochromatographic test strip is arranged in the center of a bottom plate, and a combination pad, a nitrocellulose membrane and absorbent paper are symmetrically adhered to two sides of the bottom plate in sequence. Different from a one-way detection test strip, gold particles coupled with ochratoxin A and vomitoxin antibodies are respectively fixed on the combination pads at the two ends of the two-flux test strip, and up-conversion luminescent nanoparticles coupled with the ochratoxin A and vomitoxin antigens are fixed on the corresponding T line. The invention can realize high-sensitivity and rapid detection of two detection substances. The method is simple and convenient to operate, low in cost, low in detection background value, stable in signal and high in sensitivity.

Description

High-sensitivity double-flux detection immunochromatographic test strip based on double-excitation orthogonal emission up-conversion luminescent nanoparticles
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 luminescent nanoparticles.
Background
With the growth of intensive agriculture and food industry, food safety is gradually becoming a global issue; the health reliability of food arriving in the hands of the consumer, particularly in terms of contaminant screening, should be determined in a fast, economical, accurate manner. In order to meet the demand for rapid, on-site analysis, the development of biosensors for food quality control has received increasing attention.
Ochratoxin A and vomitoxin become main factors which pollute grains in China and are byproducts, and have synergistic effect with various toxins. The mycotoxins are widely existed in the nature, wherein wheat and corn are the most polluted crops, and after being ingested by human and livestock, even trace amount of the mycotoxins have strong nephrotoxicity, mutagenicity, teratogenicity and carcinogenicity, the toxins reduce the overall immune function by reducing the immune proliferation of lymphoid organs, influence humoral immune response and generate negative feedback effect on cellular immunity, and due to the high stability of the two mycotoxins, the existing targeted detoxification method proves that the pollution and harm can not be effectively removed, so that the basic research strength must be increased, and a more effective detection method is developed to eliminate the harmfulness of the mycotoxins from the source.
The immunoassay method plays a crucial role in the current rapid screening of mycotoxins due to simplicity, feasibility and high specificity. Compared with an ELISA method, the immunoassay method based on the colloidal gold nanoparticles (AuNP) is simple and rapid in sample preparation method, results can be obtained within 3-10 min, tedious sample pretreatment, trained personnel and expensive and precise instruments are not needed, and the immunoassay method is more suitable for rapid field detection.
Upconversion nanoparticles are inorganic nanocrystals that convert near infrared light into shorter wavelength emission. This unique photophysical property, coupled with narrow emission bandwidth and large anti-stokes shift, makes UCNPs excellent optical labels for biosensing, as they can be detected without interference from the optical background of the sample matrix, receiving increasing attention in improving detection performance.
Therefore, UCNPs are combined with the traditional colloidal gold immunochromatography, a double-flux immunochromatography test strip based on a UCNP-Au composite system is developed through the arrangement of a bidirectional T line, the simultaneous detection of ochratoxin 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 immunochromatographic test strip based on double-excitation orthogonal emission up-conversion luminescent nanoparticles. The up-conversion luminescent nano-particles can simultaneously emit any one waveband of red light or green light under the excitation of two different near-infrared lights, namely, one near-infrared excitation light can excite the light of one waveband, and the near-infrared excitation light is combined with the colloidal gold, so that a brand-new double-flux detection mode is created by utilizing the bidirectional diffusion characteristic of liquid drops to realize the detection of multiple substances in food. The invention can realize single-line detection of various detection substances, and has the advantages of simple and convenient operation, low cost and higher sensitivity.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a preparation method of dual-excitation orthogonal emission up-conversion luminescent nanoparticles is characterized by comprising the following steps:
(1) The double-excitation orthogonal emission up-conversion luminescence nano-particles with a three-layer core-shell structure are prepared by adopting a seed crystal method, and NaYF doped with Yb, tm and Er 4 The nano-particles are cores, and Yb doped NaYF is wrapped outside the cores 4 A first shell layer, wherein Yb and Nd doped NaYF is wrapped outside the first shell layer 4 The second shell layer forms the Yb, tm, er and Nd co-doped NaYF 4 :Yb/Tm/Er@NaYF4:Yb@NaYF 4 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 near infrared light of 980nm and green light under the excitation of near infrared light of 808 nm.
The preparation method of the dual-excitation orthogonal emission up-conversion luminescent nano-particle 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 luminescence nano-particle through physical adsorption and the like, so as to obtain the dual-excitation orthogonal emission up-conversion luminescence nano-particle marked with the antigen of the detection object.
A preparation method of gold nanoparticles is characterized by comprising the following steps:
(1) Preparing a colloidal gold solution:
0.25mL0.1M chloroauric acid solution was added to 100mL of ultrapure water, and heated under magnetic stirring under reflux to boiling. 1.5mL L1% sodium citrate solution was added quickly and refluxing continued for about 30 minutes. During which the color of the solution changed from light yellow to black and then to dark red. And cooling to room temperature, and filtering the prepared wine red gold nanoparticle sol by using a filter membrane of 0.22 mu m to obtain the gold nanoparticles.
The preparation method of the gold nanoparticle AuNPs is characterized by further comprising the following steps:
(2) Preparation of AuNPs-antibodies: the antibody of the detection object is marked on the surface of the gold nano-particle through physical adsorption and the like, so that the gold nano-particle marked with the antibody of the detection object is obtained.
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 under 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 immunochromatographic test strip based on the double-excitation orthogonal emission up-conversion luminescent nanoparticles is characterized in that a sample pad is arranged in the center of a bottom plate, and a combination pad, an NC membrane and absorbent paper are symmetrically and sequentially adhered to two sides of the bottom plate. The assembly sequence is as follows: firstly, arranging two detection tracks on a nitrocellulose membrane, scribing a processed UCNPs solution onto an NC membrane along the specific tracks to form two test lines, symmetrically assembling the NC membrane on a bottom plate, and then sequentially adhering a combination pad, a water absorption paper and a sample pad, wherein the sample pad is assembled at the center of the test strip; the combination pad is provided with gold nanoparticles, and the gold nanoparticles are marked with antibodies of an object to be detected; immobilized on the nitrocellulose membrane is an antigen that up-converts the luminescent nanoparticle-labeled detector by dual excitation orthogonal emission.
The high-sensitivity two-flux detection immunochromatographic test strip for the dual-excitation orthogonal emission up-conversion luminescent nanoparticles is characterized in that the up-conversion luminescent nanoparticles can emit any one of red light or green light wave bands under the excitation of two different near infrared lights, only two test lines of a detection object fixed with dual-excitation orthogonal emission up-conversion luminescent nanoparticle marks need to be drawn on a nitrocellulose membrane of the immunochromatographic test strip, so that a signal of one wave band of the dual-excitation orthogonal emission up-conversion luminescent nanoparticles under the near infrared excitation light is taken as a detection T line, and a signal of the other wave band can be taken as a quality control C line, and the effect of multi-two-way single line detection is achieved.
The high-sensitivity double-flux detection immunochromatography test strip for the up-conversion luminescence nanoparticles with double-excitation orthogonal emission is characterized in that a sample pad of the double-flux test strip is arranged in the center of a bottom plate, a combination pad, an NC (numerical control) membrane and water absorption paper are adhered to the two sides of the test strip in a symmetrical sequence, the test strip is different from a one-way detection test strip, gold particles coupled with ochratoxin A and vomitoxin antibodies are fixed on the combination pads at the two ends of the double-flux test strip respectively, and UCNPs coupled with the ochratoxin A and vomitoxin antigens are fixed on corresponding T lines.
The high-sensitivity double-flux detection immunochromatography test strip for the double-excitation orthogonal emission up-conversion luminescent nanoparticles is characterized in that: the size of the commercial nitrocellulose membrane is 60cm; the combination pad is two glass fibers with the length of 5mm, the sample pad is 10mm in length, and the sample pad is stuck in the middle of the nitrocellulose membrane during assembly.
The detection method of the double-excitation double-emission fluorescence quenching colloidal gold immunochromatographic test strip for the up-conversion luminescent nanoparticles is characterized in that:
(1) When the prepared chromatographic test strip does not drop a sample to be detected on the bonding pad, antigens of a detection object marked by double-excitation orthogonal emission up-conversion luminescent nanoparticles are fixed on the nitrocellulose membrane through excitation of 980nm laser and 808nm laser respectively, and two different detection signals are obtained.
(2) When the sample does not contain a detection substance, the sample is dripped on the combination pad, and the gold nanoparticles can completely flow into the nitrocellulose membrane and are combined with the antigen of the detection substance marked by the double-excitation orthogonal emission up-conversion luminescence nanoparticles fixed on the membrane. The absorption region (520 nm) of the gold nanoparticles is just coincided with the green light emission region (540 nm) of the upconversion luminescent nanoparticles under the laser excitation of 808nm, so that the gold nanoparticles can quench green light of the upconversion luminescent nanoparticles; but the red light emitting area (660 nm) of the up-conversion luminescent nano-particles is not affected at the moment, so that a red light signal under the excitation of 980nm laser 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 corresponding antibody of the object to be detected marked on the gold nanoparticles, the detection sample and a part of the gold nanoparticles form an agglomerate which is remained on the combination pad, and the other part of the gold nanoparticles and the rest of the detection sample flow into the nitrocellulose membrane from two sides together and are combined with the antigen of the detection object marked by the double-excitation orthogonal emission up-conversion luminescence nanoparticles fixed on the membrane. Because only a part of the gold nanoparticles is combined with the antigen of the detection object marked by the dual-excitation orthogonal emission up-conversion luminescent nanoparticles fixed on the nitrocellulose membrane, the gold nanoparticles cannot completely quench the green light of the up-conversion luminescent nanoparticles, and the quenching effect is gradually reduced along with the increase of the concentration of the detection object, namely the green light signal is gradually increased. The green signal under the excitation of the 808nm laser can be used as a detection T line of the detection sample. And (3) obtaining the fluorescence intensity of the corresponding detection T line and the quality detection C line at two sides by 980nm and 808nm laser excitation, establishing the ratio of the fluorescence intensity of the T line to the fluorescence intensity of the C line, and comparing the ratio with a standard curve to realize the quantitative detection of the two components contained in the sample to be detected.
The invention has the following beneficial effects:
1. the double-flux detection immunochromatographic test strip provided by the invention only comprises two detection lines, has a simple structure, is a novel immunochromatographic test strip capable of quickly detecting two mycotoxins, mainly utilizes two signals of double-excitation orthogonal emission up-conversion luminescent nanoparticles, and can realize single-line detection of two detection substances after being combined with colloidal gold; and (3) stability of the test strip: 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 is simple in process and easy to implement; the double-excitation double-emission up-conversion luminescent nano-particles excite red light at 980nm and excite green light at 808 nm; the particle size is small: d is approximately equal to 30nm; good dispersity: PDI <0.3.
3. The detection method of the double flux detection immunochromatographic test strip takes UCNPs as biomarker antibodies, can directly observe the luminous results of two T lines under the excitation of near infrared light, eliminates background interference due to the unique optical characteristics of the UCNPs, and has the advantages of low background value, stable signal and greatly improved sensitivity; and UCNPs in the test paper are harmless to testers and the environment, and the safety is good.
4. The detection method of the double-flux detection immunochromatographic test strip does not need to carry out more pretreatment on a sample to be detected, can directly realize quantitative measurement by virtue of the up-conversion luminescence sensor, is simple and quick to operate, can be operated on site, can realize quick detection within 3-10 min, and has high result accuracy.
Drawings
FIG. 1 is a transmission electron micrograph of a dual-excitation orthogonal emission up-conversion nanoparticle;
FIG. 2 is a reaction scheme of an immune response;
FIG. 3 is a schematic diagram of the detection principle of a highly sensitive double flux detection immunochromatographic test strip based on double-excitation orthogonal emission up-conversion luminescent nanoparticles.
FIG. 4 is a schematic diagram of a detection result of a high-sensitivity double flux detection immunochromatographic test strip based on double-excitation orthogonal emission up-conversion luminescent nanoparticles.
FIG. 5 is a schematic structural diagram of a highly sensitive double flux detection immunochromatographic test strip based on double-excitation orthogonal emission up-conversion luminescent nanoparticles.
Detailed Description
Referring to fig. 1 to 5, the technical solution of the present invention is explained in detail by embodiments 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 for exciting red light at 980nm and exciting green light at 808nm, and then combining with colloidal gold to prepare a dual-flux detection immunochromatography test strip, and simultaneously detecting ochratoxin A and vomitoxin, and comprises the following steps:
1. preparation of UCNPs
1.1 preparing double-excitation orthogonal emission up-conversion luminescence nanoparticles with a three-layer core-shell structure by adopting a seed crystal method, wherein NaYF doped with Yb, tm and Er is used 4 The nano-particles are cores, and Yb doped NaYF is wrapped outside the cores 4 A first shell layer, and Yb and Nd doped NaYF is wrapped outside the first shell layer 4 A second shell layer which forms Yb, tm, er and Nd co-doped NaYF 4 :Yb/Tm/Er@NaYF4:Yb@NaYF 4 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. synthesis of NaYF doped with Yb, tm and Er 4 Nanoparticles as inner core
10mL of Oleic Acid (OA), 10mL of Octadecene (ODE), 0.84g of NaF solid, and Yb (OAc) were weighed out 3 0.0683g、Tm(OAc) 3 0.0017g、Er(OAc) 3 0.2752g of the mixture is added into a three-neck flask A, the mixture is magnetically stirred and heated to 110-120 ℃, the mixture is kept for 10min, and then the mixture is vacuumized to remove water and oxygen; removing and then introducing N 2 Heating to 300 ℃ and reacting for 1h;
b. wrapping Yb doped NaYF outside the inner core 4 First shell layer
Weighing Oleic Acid (OA) 4mL, octadecene (ODE) 4mL, naF solid 0.4788g, yb (OAc) 3 0.07g of the mixture is added into a three-neck flask B, the mixture is magnetically stirred and heated to 110 to 120 ℃, the mixture is kept for 10min, and then the mixture is vacuumized to remove water and oxygen; removing all and then introducing N 2 Heating to 150 ℃, injecting the mixture into the three-neck flask A at the speed of 0.13mL/min by using a needle tube after the reaction in the step a is finished, and reacting for 1h at the temperature of 300 ℃;
c. a second shell layer of Yb and Nd is wrapped outside the first shell layer
Weighing 4mL Oleic Acid (OA), 4mL Octadecene (ODE), and Yb (OAc) 3 0.0525g、Nd(OAc) 3 0.4333g, adding into a three-neck flask C, magnetically stirring, heating to 110-120 ℃, keeping for 10min, and then vacuumizing to remove water and oxygen; removing all and then introducing N 2 After the reaction in the step b is finished, injecting the mixture into the three-neck flask A by using a needle tube at the speed of 0.13mL/min, and reacting for 100min at 300 ℃; after the reaction is finished, cooling to room temperature, putting the reaction liquid in the three-neck flask A into a centrifugal tube, and centrifugally separating out the obtained nano particles;
d. stripping oleic acid from the surface of the nanoparticles obtained in step c
And (c) taking the nanoparticles obtained in the step (c), adding a mixed solution of ethanol with pH =1 and concentrated hydrochloric acid (7.5 mL of ethanol and 62.5 mu L of concentrated hydrochloric acid), performing ultrasonic dispersion uniformly, performing vibration while performing ultrasonic treatment for 30min, centrifuging, removing a supernatant, adding a mixed solution of ethanol with pH =4 and concentrated hydrochloric acid (7.5 mL of ethanol and 7.5mL of concentrated hydrochloric acid) into the nanoparticles, performing ultrasonic dispersion uniformly, performing vibration while performing ultrasonic treatment for 30min, performing centrifugal separation again, and washing the nanoparticles with water to obtain the water-soluble dual-excitation orthogonal emission up-conversion luminescent nanoparticles UCNPs.
2. Preparation of UCNPs-antigens
Adding 0.02moL/L K into water-soluble double-excitation orthogonal emission up-conversion luminescence nanoparticles with the concentration of 1mg/mL 2 CO 3 And (4) adjusting the pH of the mixed solution to about 8.5. 10. Mu.g of AFB1 antigen was added to the mixed solution, mixed uniformly for 30 seconds, and incubated in a shaker at 37 ℃ for 23min with shaking. Then, 200. Mu.L of BSA (10% by mass) solution and 200. Mu.L of PEG 20000 (1% by mass) solution were added to the mixed solution, and incubation with shaking at 110 ℃ for 15min was continued in a shaker. The mixed solution was placed in a centrifuge tube, centrifuged at 13000r/min at 4 ℃ for 30min, and redispersed in 1% bsa in 0.01M, pH =7.0 PBS buffer, to complete the preparation of the solution of the dual excitation orthogonal emission up-conversion luminescent nanoparticles labeled with the antigen of AFB1 to be detected.
3. Preparing a colloidal gold solution:
0.25mL0.1M chloroauric acid solution was added to 100mL of ultrapure water, and heated under magnetic stirring to reflux to boiling. 1.5ml1% sodium citrate solution was added quickly and refluxing continued for about 30 minutes. During which the color of the solution changed from light yellow to black and then to dark red. And cooling to room temperature, and filtering the prepared wine red gold nanoparticle sol by using a filter membrane of 0.22 mu m to obtain the gold nanoparticles.
4. Preparation of AuNPs antibodies
10mL of the prepared gold nanoparticles were collected and concentrated to 2mL by centrifugation. Adding 0.02moL/L of K into gold nanoparticles with the concentration of 1mg/mL 2 CO 3 And (4) adjusting the pH of the mixed solution to about 8.5.10 μ g of AFB1 antibody was added to the mixed solution, mixed uniformly for 30 seconds, and incubated with shaking in a shaker at 110 ℃ for 20min. To the mixed solution, 200. Mu.L of BSA (10% by mass) solution and 200. Mu.L of PEG 20000 (1% by mass) solution were added, and incubation with shaking at 37 ℃ for 15min was continued in a shaker. The mixed solution was put into a centrifuge tube, centrifuged at 13000r/min at 4 ℃ for 30min, and redispersed in a PBS buffer solution containing 1% bsa at 0.01M and pH =7.0, thereby completing the preparation of the solution of the gold nanoparticles labeled with the antibody to AFB1 to be detected.
5. The preparation of the chromatographic test strip has a specific structure shown in figure 5.
Two round glass fibers (conjugate pads) 5mm wide were immersed in the antibody-Au NP solution at a concentration of 4mg/mL for 10 minutes, and then dried at 37 ℃ for 2 hours. Antigen (OTA-BSA, DON-BSA) coupled UCNPs solution was diluted to 4mg/mL and streaked separately along specific distances at a concentration of 1.5. Mu.L/cm onto NC membranes 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 size of the commercial nitrocellulose membrane is 60cm; the combination pad is 5 mm's width circular glass fiber, and the diameter of sample pad is 10mm, and the sample pad glues in the intermediate position of test paper strip during the equipment.
The high-sensitivity double-flux detection immunochromatographic test strip based on double-excitation orthogonal emission up-conversion luminescent nanoparticles further comprises the following steps:
(1) Treatment of bond pads
Taking a glass cellulose membrane as a bonding pad material, firstly soaking the bonding pad in a bonding pad treatment solution for 24h, then drying at 37 ℃, putting the bonding pad into the solution of the gold nanoparticle marked with the OTA and DON antibodies to be detected obtained in the step 1, soaking for 10min, and then drying at 37 ℃ to finish the treatment of the bonding pad; the conjugate pad treatment solution was 0.01M, pH =7.4 PBS buffer solution containing 1% BAS, 2% Tween-20 and 5% sucrose by mass concentration.
(2) Treatment of nitrocellulose membranes
Scribing a specific annular track of a solution of double-excitation orthogonal emission up-conversion luminescence nanoparticles for marking antigens of OTA and DON to be detected on a nitrocellulose membrane at a parameter of 1.5 muL/cm, and drying to finish the treatment of the nitrocellulose membrane;
(3) Assembly of test strips
And adhering the treated combined pad to the circle center position of each test ring on the nitrocellulose membrane to complete the assembly of the multiple double-flux test strip.
6. Method and process for detecting colloidal gold immune test paper strip
(1) When the prepared chromatographic test strip does not drop a sample to be detected on a sample pad, antigens of a detection object marked by dual-excitation orthogonal emission up-conversion luminescent nanoparticles are fixed on the cellulose nitrate membrane through laser excitation of 980nm and 808nm respectively, and two different detection signals are obtained.
(2) When the sample does not contain a detection substance, the sample is dripped on the sample pad, and the gold nanoparticles can completely flow into the nitrocellulose membrane towards two sides and are combined with the antigen of the detection substance which is fixed on the membrane and emits up-conversion luminescence nanoparticles through double excitation orthogonal emission. The absorption area (520 nm) of the gold nanoparticles is just coincided with the green light emission area (540 nm) of the upconversion luminescent nanoparticles under the excitation of 808nm laser, so that the gold nanoparticles can quench green light of the upconversion luminescent nanoparticles; but the red light emitting area (660 nm) of the up-conversion luminescent nano-particles is not affected at the moment, so that a red light signal under the excitation of 980nm laser 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 corresponding antibody of the object to be detected marked on the gold nanoparticles, the detection sample and a part of the gold nanoparticles form an agglomerate which is remained on the combination pad, and the other part of the gold nanoparticles and the rest of the detection sample flow into the nitrocellulose membrane together and are combined with the antigen of the detection object marked by the double-excitation orthogonal emission up-conversion luminescence nanoparticles fixed on the membrane. Because only a part of the gold nanoparticles is combined with the antigen of the detection object marked by the dual-excitation orthogonal emission up-conversion luminescent nanoparticles fixed on the nitrocellulose membrane, the gold nanoparticles cannot completely quench the green light of the up-conversion luminescent nanoparticles, and the quenching effect is gradually reduced along with the increase of the concentration of the detection object, namely the green light signal is gradually increased. The green signal under the excitation of the 808nm laser can be used as a detection T line of the detection sample. And (3) obtaining the fluorescence intensity of the corresponding two-side detection T line and the quality detection C line by laser excitation of 980nm and 808nm, establishing a ratio of the fluorescence intensity of the T line to the fluorescence intensity of the C line, and comparing the ratio with a standard curve to realize quantitative detection of two components (OTA and DON) contained in the sample to be detected.
The above description is only exemplary of the invention and should not be taken as limiting the invention, as any modification, equivalent replacement, or improvement made within the spirit and principle of the invention should be included in the protection scope of the invention.

Claims (11)

1. A preparation method of dual-excitation orthogonal emission up-conversion luminescent nanoparticles is characterized by comprising the following steps:
(1) The double-excitation orthogonal emission up-conversion luminescence nano-particles with a three-layer core-shell structure are prepared by adopting a seed crystal method, and NaYF doped with Yb, tm and Er 4 The nano-particles are cores, and Yb doped NaYF is wrapped outside the cores 4 A first shell layer, and Yb and Nd doped NaYF is wrapped outside the first shell layer 4 A second shell layer which forms Yb, tm, er and Nd co-doped NaYF 4 :Yb/Tm/Er@NaYF4:Yb@NaYF 4 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 near infrared light of 980nm and green light under the excitation of near infrared light of 808 nm.
2. The method for preparing dual-excitation orthogonal emission up-conversion luminescent nanoparticles (UCNPs) according to claim 1, 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 luminescence nano-particle through physical adsorption and the like, so as to obtain the dual-excitation orthogonal emission up-conversion luminescence nano-particle marked with the antigen of the detection object.
3. A preparation method of gold nanoparticles is characterized by comprising the following steps:
(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 under reflux to boiling. 1.5mL of a 1% solution of sodium citrate was added quickly and refluxing continued for about 30 minutes. During which the color of the solution changed from light yellow to black and then to dark red. And cooling to room temperature, and filtering the prepared wine red gold nanoparticle sol by using a filter membrane of 0.22 mu m to obtain the gold nanoparticles.
4. The method for preparing gold nanoparticle AuNPs as claimed in claim 4, further comprising the steps of:
(2) Preparation of AuNPs-antibodies: the antibody of the detection object is marked on the surface of the gold nano-particle through physical adsorption and the like, so that the gold nano-particle marked with the antibody of the detection object is obtained.
5. The dual-excitation orthogonal emission upconversion luminescent nanoparticle prepared according to the method of any one of claims 1 to 4, wherein the upconversion luminescent nanoparticle can emit light of either red or green wavelength band simultaneously under excitation of two different near infrared lights, i.e. one near infrared excitation light can excite light of one wavelength band.
6. A high-sensitivity double-flux detection immunochromatography test strip prepared by using the double-excitation orthogonal emission up-conversion luminescence nanoparticles as claimed in claim 5, wherein a sample pad of the double-flux test strip is arranged in the center of a bottom plate, a binding pad, a nitrocellulose membrane and absorbent paper are adhered to the two sides of the bottom plate in a symmetrical sequence, different from a one-way 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-flux test strip, double-excitation orthogonal emission up-conversion nanoparticles coupled with ochratoxin A and vomitoxin antigens are fixed on corresponding T lines, and when a sample to be detected is dripped, a solution to be detected migrates to the two sides at the same speed.
7. A high-sensitivity double-flux detection immunochromatographic test strip based on double-excitation orthogonal emission up-conversion luminescent nanoparticles, which is characterized in that the up-conversion luminescent nanoparticles can emit any one of red light or green light under the excitation of near infrared light of two different wave bands simultaneously, only two antigen detection line T lines of a detection object fixed with a mark of the double-excitation orthogonal emission up-conversion luminescent nanoparticles need to be coated on a nitrocellulose membrane of the immunochromatographic test strip in a double-direction mode, so that a signal of one wave band emitted by the double-excitation orthogonal emission up-conversion luminescent nanoparticles under the near infrared excitation light is taken as a detection T line, and a signal of the other wave band is taken as a quality control C line, thereby achieving the effect of double-direction single-line detection.
8. The high-sensitivity double-flux detection immunochromatographic test strip based on double-excitation orthogonal emission up-conversion luminescence nanoparticles as claimed in claim 6, wherein a sample pad of the double-flux test strip is arranged in the center of a bottom plate, a binding pad, an NC membrane and a water absorption paper are adhered in a symmetrical order on two sides, unlike a single-flux detection test strip, gold particles coupled with ochratoxin A and vomitoxin antibodies are fixed on the binding pads on two ends of the double-flux test strip respectively, and UCNPs coupled with ochratoxin A and vomitoxin antigens are fixed on corresponding T lines.
9. The highly sensitive dual flux assay immunochromatographic test strip based on dual excitation orthogonal emission up-conversion luminescent nanoparticles according to claim 6, characterized in that: the size of the commercial nitrocellulose membrane is 60cm; the combination pad is two glass fibers with the length of 5mm, the sample pad is 10mm in length, and the sample pad is stuck in the middle of the nitrocellulose membrane during assembly.
10. The detection method of the high-sensitivity double-flux detection immunochromatographic test strip based on the double-excitation orthogonal emission up-conversion luminescent nanoparticles according to one of claims 8 to 10, which is characterized in that:
(1) When the prepared immunochromatography test strip is not used for dropwise adding a sample to be detected in the center of the binding pad, antigens of a detection object marked by double-excitation orthogonal emission up-conversion luminescent nanoparticles are fixed on the nitrocellulose membrane through excitation of 980nm laser and 808nm laser respectively, and two different detection signals are obtained.
(2) When the sample does not contain the detection substance, the sample is dripped in the center of the sample pad, and the gold nanoparticles can completely flow into two sides of the nitrocellulose membrane and are combined with the antigen of the detection substance marked by the double-excitation orthogonal emission up-conversion luminescent nanoparticles fixed on the membrane. The absorption area (520 nm) of the gold nanoparticles is just coincided with the green light emission area (540 nm) of the upconversion luminescent nanoparticles under the excitation of 808nm laser, so that the gold nanoparticles can quench green light of the upconversion luminescent nanoparticles; but at the moment, the red light emitting region (660 nm) of the up-conversion luminescent nanoparticle is not influenced, so that a red light signal under the excitation of 980nm laser can be used as a quality control C line of a detection sample.
11. The method for detecting the high-sensitivity double-flux detection immunochromatographic strip based on the double-excitation orthogonal emission up-conversion luminescent nanoparticle according to claim 10, wherein 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 corresponding antibody of the object to be detected marked on the gold nano particles on the two sides, the detection sample and a part of the gold nano particles form an agglomerate which is remained 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 on the dual-excitation orthogonal emission up-conversion luminescence nano particles fixed on the membrane. Because only a part of the gold nanoparticles is combined with the antigen of the detection object marked by the dual-excitation orthogonal emission up-conversion luminescent nanoparticles fixed on the nitrocellulose membrane, the gold nanoparticles cannot completely quench the green light of the up-conversion luminescent nanoparticles, and the quenching effect is gradually reduced along with the increase of the concentration of the detection object, namely the green light signal is gradually increased. At this time, a green light signal under the excitation of the 808nm laser can be used as a detection T line of the detection sample. And (3) obtaining the fluorescence intensity of the corresponding two-side detection T line and the quality detection C line by laser excitation of 980nm and 808nm, establishing a ratio of the fluorescence intensity of the T line to the fluorescence intensity of the C line, and comparing the ratio with a standard curve to realize quantitative detection of two components contained in the sample to be detected.
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