CN111303878A - Up-conversion luminescent nanoparticle preparation and chromatography test strip based on double excitation and double emission and detection method - Google Patents

Up-conversion luminescent nanoparticle preparation and chromatography test strip based on double excitation and double emission and detection method Download PDF

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CN111303878A
CN111303878A CN201910299813.8A CN201910299813A CN111303878A CN 111303878 A CN111303878 A CN 111303878A CN 201910299813 A CN201910299813 A CN 201910299813A CN 111303878 A CN111303878 A CN 111303878A
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张勇
刘金亮
袁赟
张菁
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Beijing Transpacific Technology Development Ltd
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Abstract

The invention provides a double-excitation double-emission based up-conversion luminescence nanoparticle preparation and chromatography test strip and a detection method. The up-conversion nano particles can emit any two wave bands of red light, green light or blue light under the excitation of two different near-infrared lights, namely, one near-infrared excitation light can excite the light of one wave band. The chromatography test strip consists of a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a bottom plate, wherein the nitrocellulose membrane is provided with a detection line and a quality control line; an antibody of the detection object marked by the double-excitation double-emission up-conversion luminescent nano particles is fixed on the bonding pad. The invention can detect the substances of two components at the same time, and can realize double authentication on the detection object of single component. And realizing qualitative detection of a detection object contained in the sample to be detected according to the color change of the converted nanoparticles on the detection area of the test strip. The method and the test paper have the advantages of simple and convenient operation, low detection background value, stable signal and higher sensitivity.

Description

Up-conversion luminescent nanoparticle preparation and chromatography test strip based on double excitation and double emission and detection method
Technical Field
The invention belongs to the technical field of biomedical diagnosis, and particularly relates to a double-excitation double-emission based up-conversion luminescence nanoparticle preparation and chromatography test strip and a detection method.
Background
Harmful chemical and biological contaminants in environmental and food samples are a global concern due to adverse effects on the ecosystem. These substances originate from residual medical (pharmaceutical pollutants), agricultural (pesticides), biological (pathogens and their released toxins) and industrial (chemical solvents, industrial by-products, etc.) waste, are extremely carcinogenic and have a negative impact on human health. Therefore, the timely detection of carcinogens in food has become a major issue in modern biomedicine.
The immunochromatographic test strip is based on capillary force, and qualitatively or quantitatively analyzes a target detection object by strip color development through antigen-antibody specific immunoreaction. Upconversion fluorescent nanoparticles are nanoparticles that convert excitation light of a long wavelength (e.g., near infrared light) into emission light of a short wavelength (e.g., ultraviolet light, visible light, etc.), and different absorption and emission spectra are generally obtained by the difference in composition of the particles. Compared with other nanoparticles, the up-conversion fluorescent nanoparticles have low toxicity, high sensitivity and strong stability, and are less interfered by background signals, so that the UCNPs are combined with the immunochromatographic test paper, the self-luminous interference phenomenon of a biological sample can be eliminated, the signal-to-noise ratio is improved, the sensitivity and the stability are enhanced, and the high-sensitivity quantitative detection is realized. Thus, the upconversion fluorescent nanoparticles become one of the best choices for in vitro detection.
The up-conversion test paper has the advantages of quick detection, easy operation, portability and low cost, and the detected carcinogens are common:
1. aflatoxin (AFT)
The aflatoxin is a secondary metabolite with similar structure and the same toxic group, and is mainly a toxin produced by fungi such as aspergillus flavus, aspergillus parasiticus and the like. Aflatoxin has carcinogenic, teratogenic, and mutagenic reproductive toxicity, wherein aflatoxin AFB1 has the greatest toxicity and harm.
However, when the existing upconversion test strip is used for detecting carcinogens, the nanoparticles for labeling the carcinogen antibodies only have one detection signal, so that the quantitative detection result of the carcinogen concentration in the sample to be detected remains uncertain.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a preparation method of up-conversion luminescent nanoparticles based on dual excitation and dual emission and conversion luminescent nanoparticles, so that the up-conversion luminescent nanoparticles have two signals, and can emit any two wave bands of red light, green light or blue light under the excitation of two different near infrared lights, namely one near infrared excitation light can excite the light of one wave band.
The invention also provides a chromatography test strip and a detection method adopting the double-excitation double-emission up-conversion luminescence nanoparticle, which can simultaneously detect two components of objects to be detected, can realize double authentication on a single component of objects to be detected, and improve the accuracy of quantitative detection. .
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a preparation method of double-excitation double-emission up-conversion nanoparticles is characterized by comprising the following steps:
(1) the method adopts a seed crystal method to prepare double-excitation double-emission up-conversion nanoparticles with a three-layer core-shell structure, and NaYF doped with Yb, Tm and Er4The nano-particles are cores, and Yb doped NaYF is wrapped outside the cores4A first shell layer, and Yb and Nd doped NaYF is wrapped outside the first shell layer4A second shell layer which forms Yb, Tm, Er and Nd co-doped NaYF4:Yb/Tm/Er@NaYF4:Yb@NaYF4Yb/Nd core-shell structure; the water-soluble dual-excitation dual-emission up-conversion nano-particle UCNPs are prepared, and the up-conversion nano-particle emits 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 dual-emission up-conversion nanoparticle UCNPs is characterized in that the step (1) specifically comprises the following steps:
a. synthesis of NaYF doped with Yb, Tm and Er4Made of nanoparticlesIs a kernel
10mL of Oleic Acid (OA), 10mL of Octadecene (ODE), 0.84g of NaF solid, and Yb (OAc) were weighed out30.0683g、Tm(OAc)30.0017g、Er(OAc)30.2752g, adding into a three-neck flask A, magnetically stirring, heating to 110-120 ℃, keeping for 10min, and then vacuumizing to remove water and oxygen; removing all and then introducing N2Heating to 300 ℃ and reacting for 1 h;
b. wrapping Yb doped NaYF outside the inner core4First shell
4mL of Oleic Acid (OA), 4mL of Octadecene (ODE), 0.4788g of NaF solid, and Yb (OAc) were weighed out30.07g 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 N2Heating 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)30.0525g、Nd(OAc)30.4333g, 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 N2After 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 ℃; cooling to room temperature after the reaction is finished, putting the reaction liquid in the three-neck flask A into a centrifugal tube, and centrifugally separating to obtain the obtained nano particles;
d. stripping oleic acid from the surface of the nanoparticles obtained in step c
And c, taking the nano-particles obtained in the step c, adding a mixed solution of ethanol with the pH value of 1 and concentrated hydrochloric acid (7.5mL of ethanol and 62.5 mu L of concentrated hydrochloric acid), carrying out ultrasonic dispersion uniformly, then carrying out vibration while carrying out ultrasonic treatment for 30min, then centrifuging, removing a supernatant, adding a mixed solution of ethanol with the pH value of 4 and concentrated hydrochloric acid (7.5mL of ethanol and 7.5mL of concentrated hydrochloric acid), carrying out ultrasonic dispersion uniformly, carrying out vibration while carrying out ultrasonic treatment for 30min, then carrying out centrifugal separation again, and washing the obtained nano-particles with water to obtain the water-soluble dual-excitation dual-emission upconversion nano-particle UCNPs.
The preparation method of the dual-excitation dual-emission up-conversion nanoparticle UCNPs is characterized by further comprising the following steps:
(2) surface treatment of the upconversion nanoparticles: the method is characterized in that a layer of polypropylene is coated on the surface of the upconversion nanoparticles through the actions of electrostatic adsorption and the like, and specifically comprises the following steps:
taking 5mg of the double-excitation double-emission up-conversion nanoparticles prepared in the step (1), taking the ratio of the mass of the up-conversion nanoparticles to the mass of the polypropylene solution as 4:1, dispersing the up-conversion nanoparticles in 1mg/mL of polypropylene buffer solution, carrying out shaking table reaction at normal temperature for 12-14 h, then carrying out centrifugal separation at 4 ℃ to obtain the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene, dispersing the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene in PBS buffer solution containing 0.01M, pH-7.0-8.0, and storing at 4 ℃, thus completing the preparation of the solution of the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene.
The preparation method of the dual-excitation dual-emission up-conversion nanoparticle UCNPs is characterized by further comprising the following steps:
(3) preparation of UCNPs-antibodies: marking the AFB1 and AFB2 antibodies or compound antibodies thereof on the surface of the polypropylene-coated double-excitation double-emission up-conversion nanoparticles through the action of electrostatic adsorption and the like to obtain the double-excitation double-emission up-conversion nanoparticles marked with the AFB1 and AFB2 antibodies or compound antibodies thereof to be detected,
the double-excitation double-emission up-conversion nanoparticle prepared by the method is characterized in that the up-conversion nanoparticle can be used for simultaneously detecting substances containing AFB1 and AFB2, and can also be used for realizing double authentication on a detected substance of a single component of AFB1 or AFB2 to be detected; the up-conversion nano particles can emit any two wave bands of red light, green light or blue light under the excitation of two different near infrared light.
The chromatography test strip adopting the double-excitation double-emission up-conversion nanoparticles is characterized by comprising a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially arranged on the bottom plate from left to right; wherein the nitrocellulose membrane is arranged in the middle of the bottom plate, the sample pad is arranged on the left side of the nitrocellulose membrane, and a combination pad is arranged between the nitrocellulose membrane and the sample pad; the absorbent paper is arranged on the right side of the nitrocellulose membrane; wherein, the sample pad and the combination pad, the combination pad and the nitrocellulose membrane, and the nitrocellulose membrane and the absorbent paper are partially laminated with each other; the binding pad is provided with double-excitation double-emission up-conversion nanoparticles, and antibodies of AFB1 and/or AFB2 to be detected are/is marked on the up-conversion nanoparticles; one or two detection T lines are arranged in different areas on the nitrocellulose membrane, wherein T1 is coated with AFB1 antigen, and T2 is coated with AFB2 antigen; and a quality control C line is arranged at the same time, and a goat anti-mouse IgG antibody is coated on the quality control C line.
The chromatography test strip of the double-excitation double-emission up-conversion nanoparticles is characterized in that the bottom plate is a viscous plastic bottom plate, and viscous substances are arranged on the upper surface of the bottom plate; the assembly sequence is as follows: and sequentially adhering the treated sample pad, the bonded combination pad, the bonded nitrocellulose membrane and the absorbent paper from the left side to the right side of the adhesive plastic base plate.
The chromatography test strip for the double-excitation double-emission up-conversion nanoparticles is characterized in that: the length of the viscous plastic bottom plate is 60 mm; the length of the sample pad, the length of the combination pad, the length of the nitrocellulose membrane and the length of the absorbent paper are 8-17 mm, and contact areas of all parts are mutually overlapped and pressed for 1-3 mm during assembly.
The detection method of the chromatography test strip of the double-excitation double-emission up-conversion nanoparticles is characterized in that qualitative detection of a detection object contained in a sample to be detected is realized according to color change of the up-conversion nanoparticles in a test strip detection area, and the detection method specifically comprises the following steps:
(1) dropwise adding a test sample to be detected on the sample pad, wherein the test sample to be detected can be combined with the corresponding antibody of the substance to be detected marked on the up-conversion nanoparticles, and flows into the nitrocellulose membrane together, and part of the nitrocellulose membrane is fixed on the T line to be detected, and the rest of the nitrocellulose membrane flows through the quality control C line along with the test sample to be detected;
(2) when detecting a single-component substance, firstly obtaining detection signals of a T line and a C line after being excited by 980nm laser, then obtaining detection signals of the T line and the C line after being excited by 808nm laser, and authenticating the detection signals of the former wave band by using the detection signals of the latter wave band;
(3) when the two components are detected simultaneously, detection signals of two different T lines and C lines are obtained by respectively exciting the two components by 980nm laser and 808nm laser, and one component in a sample to be detected is judged according to the difference of the detection signals, so that qualitative detection for simultaneously detecting the two components in the sample to be detected is realized.
The step (1) further comprises the following steps:
(11) before detection, solutions of detection objects with different concentrations are respectively dripped on a sample pad for detection; and (3) obtaining the fluorescence intensity of the corresponding detection T line and the fluorescence intensity of the corresponding quality detection C line through laser excitation of 980nm and 808nm, 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 quantitative detection of the components contained in the sample to be detected.
The invention has the following beneficial effects:
1. the preparation method of the up-conversion nano-particles based on double excitation and double emission, which is provided by the invention, has the advantages of simple process and easy implementation; the double-excitation double-emission up-conversion nanoparticles excite 980nm red light and 808nm green light;
2. the chromatographic test strip provided by the invention has a compact structure, is a novel immune test strip capable of rapidly detecting carcinogens, and mainly utilizes that double-excitation double-emission up-conversion nanoparticles have two signals, so that substances of two components can be detected simultaneously, double authentication can be realized on a single-component detection object, and the accuracy of quantitative detection is improved.
3. According to the detection method of the chromatographic test strip, the UCNPs are used as biomarker antibodies, the result can be directly observed under the excitation of near infrared light, due to the unique optical characteristics of the UCNPs, background interference is eliminated, 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 the safety is good.
4. The chromatography test strip level detection method provided by the invention has the advantages that the sample to be detected does not need to be subjected to more pretreatment, quantitative measurement can be directly realized by virtue of the up-conversion luminescence sensor, the operation is simple and rapid, the field operation can be realized, and the result accuracy is high.
Drawings
FIG. 1 is a schematic structural diagram of an upconversion test strip;
FIG. 2 is a schematic diagram of the reaction for detecting a single component immune response;
FIG. 3 is a reaction scheme for simultaneously detecting two component immune responses.
Detailed Description
The technical solution of the present invention will be explained in detail below with reference to examples and drawings.
Example 1:
please refer to fig. 1 and fig. 2.
The preparation method of the dual-excitation dual-emission up-conversion nanoparticle provided by this embodiment is used to prepare a dual-excitation dual-emission up-conversion nanoparticle that excites red light at 980nm and excites green light at 808nm, and then prepare an analytical test strip, and detect a single component of AFB1 (one type of AFT), and includes the following steps:
(1) the method adopts a seed crystal method to prepare double-excitation double-emission up-conversion nanoparticles with a three-layer core-shell structure, and NaYF doped with Yb, Tm and Er4The nano-particles are cores, and Yb doped NaYF is wrapped outside the cores4A first shell layer, and Yb and Nd doped NaYF is wrapped outside the first shell layer4A second shell layer which forms Yb, Tm, Er and Nd co-doped NaYF4:Yb/Tm/Er@NaYF4:Yb@NaYF4Yb/Nd core-shell structure; the water-soluble dual-excitation dual-emission up-conversion nano-particle UCNPs are prepared, and the up-conversion nano-particle emits 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 step (1) specifically comprises the following steps:
a. synthesis of NaYF doped with Yb, Tm and Er4Nanoparticles as inner core
10mL of Oleic Acid (OA), 10mL of Octadecene (ODE), 0.84g of NaF solid, and Yb (OAc) were weighed out30.0683g、Tm(OAc)30.0017g、Er(OAc)30.2752g, adding into a three-neck flask A, magnetically stirring, heating to 110-120 ℃, keeping for 10min, and then vacuumizing to remove water and oxygen; removing all and then introducing N2Heating to 300 ℃ and reacting for 1 h;
b. wrapping Yb doped NaYF outside the inner core4First shell
4mL of Oleic Acid (OA), 4mL of Octadecene (ODE), 0.4788g of NaF solid, and Yb (OAc) were weighed out30.07g 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 N2Heating 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)30.0525g、Nd(OAc)30.4333g, 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 N2After 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 ℃; cooling to room temperature after the reaction is finished, putting the reaction liquid in the three-neck flask A into a centrifugal tube, and centrifugally separating to obtain the obtained nano particles;
d. stripping oleic acid from the surface of the nanoparticles obtained in step c
And c, taking the nano-particles obtained in the step c, adding a mixed solution of ethanol with the pH value of 1 and concentrated hydrochloric acid (7.5mL of ethanol and 62.5 mu L of concentrated hydrochloric acid), carrying out ultrasonic dispersion uniformly, then carrying out vibration while carrying out ultrasonic treatment for 30min, then centrifuging, removing a supernatant, adding a mixed solution of ethanol with the pH value of 4 and concentrated hydrochloric acid (7.5mL of ethanol and 7.5mL of concentrated hydrochloric acid), carrying out ultrasonic dispersion uniformly, carrying out vibration while carrying out ultrasonic treatment for 30min, then carrying out centrifugal separation again, and washing the obtained nano-particles with water to obtain the water-soluble dual-excitation dual-emission upconversion nano-particle UCNPs.
(2) Surface treatment of the upconversion nanoparticles: the method is characterized in that a layer of polypropylene is coated on the surface of the upconversion nanoparticles through the actions of electrostatic adsorption and the like, and specifically comprises the following steps:
taking 5mg of the double-excitation double-emission up-conversion nanoparticles prepared in the step (1), taking the ratio of the mass of the up-conversion nanoparticles to the mass of the polypropylene solution as 4:1, dispersing the up-conversion nanoparticles in 1mg/mL of polypropylene buffer solution, carrying out shaking table reaction at normal temperature for 12-14 h, then carrying out centrifugal separation at 4 ℃ to obtain the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene, dispersing the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene in PBS buffer solution containing 0.01M, pH-7.0-8.0, and storing at 4 ℃, thus completing the preparation of the solution of the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene.
(3) Preparation of UCNPs-antibodies: the surface of the double-excitation double-emission up-conversion nano-particle coated with the polystyrene is labeled with an antibody of AFB1 through the action of electrostatic adsorption and the like.
Adding EDC and NHS into a solution of double-excitation double-emission upconversion nanoparticles wrapped with polypropylene according to the carboxyl ratio of the upconversion nanoparticles to EDC of 1:10 and the ratio of EDC to NHS of about 1:5, carrying out shaking table reaction for 30min at normal temperature, and carrying out centrifugal separation at 4 ℃ to activate the upconversion nanoparticles. Adding the antibody of AFB1 to be detected into a solution of double-excitation double-emission up-conversion nanoparticles wrapping polypropylene according to the mass ratio of the up-conversion nanoparticles to the antibody of the carcinogen to be detected of 100-300: 1, carrying out shaking table reaction for 4 hours at normal temperature, carrying out centrifugal separation at 4 ℃ to obtain double-excitation double-emission up-conversion nanoparticles labeling the antibody of AFB1 to be detected, and carrying out storage at 4 ℃ in PBS (phosphate buffered saline) buffer solution containing 50mM glycine and 0.01-3% Tween-20, wherein 0.01M, pH is 7.0-8.0, thus completing preparation of the solution of the double-excitation double-emission up-conversion nanoparticles labeling the antibody of AFB1 to be detected.
Referring to fig. 1(a) and fig. 2, a chromatography test strip using the dual-excitation dual-emission upconversion nanoparticles comprises a bottom plate 1, and a sample pad 2, a binding pad 3, a nitrocellulose membrane 5 and absorbent paper 4 which are sequentially arranged on the bottom plate 1 from left to right; wherein the nitrocellulose membrane 5 is arranged in the middle of the bottom plate 1, the sample pad 2 is arranged on the left side of the nitrocellulose membrane 5, and the combination pad 3 is arranged between the nitrocellulose membrane and the sample pad; the absorbent paper 4 is arranged on the right side of the nitrocellulose membrane 5; wherein, the sample pad 2 and the combination pad 3, the combination pad 3 and the nitrocellulose membrane 5, and the nitrocellulose membrane 5 and the absorbent paper 4 are partially laminated with each other; the binding pad 3 is provided with double-excitation double-emission up-conversion nanoparticles, and antibodies of AFB1 to be detected are marked on the up-conversion nanoparticles; a detection T line is arranged in different areas on the nitrocellulose membrane 5, and the T1 is coated with AFB1 antigen; and a quality control C line is arranged at the same time, and a goat anti-mouse IgG antibody is coated on the quality control C line.
The bottom plate 1 of the chromatographic test strip is a viscous plastic bottom plate, and viscous substances are arranged on the upper surface of the bottom plate; the assembly sequence is as follows: the sample pad 2 after the bonding treatment, the bonding pad 3 after the bonding treatment, the nitrocellulose membrane after the bonding treatment, and 5 are sequentially bonded from the left side to the right side of the adhesive plastic base plate 1, and finally, the absorbent paper 4 is bonded.
The length of the viscous plastic bottom plate 1 is 60 mm; the length of the sample pad 2, the length of the combination pad 3, the length of the nitrocellulose membrane 5 and the length of the absorbent paper 4 are 8-17 mm, and the overlapping length of the contact area parts is 1-3 mm.
The detection method of the chromatography test strip of the double-excitation double-emission up-conversion nanoparticles realizes the qualitative detection of the detection object contained in the sample to be detected according to the color change of the up-conversion nanoparticles in the test strip detection area, and specifically comprises the following steps:
(1) before detection, solutions of detection objects with different concentrations are respectively dripped on a sample pad for detection; the fluorescence intensity of a corresponding detection T line and a corresponding detection C line is obtained through laser excitation of 980nm and 808nm, 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 components in the sample to be detected is realized through comparison with a standard curve;
(2) dripping a test sample to be detected on the sample pad, wherein the test sample to be detected can be combined with the corresponding antibody of the substance to be detected marked on the up-conversion nano particles, and flows into the nitrocellulose membrane 5 together, and part of the nitrocellulose membrane is fixed on a detected T line, and the rest of the nitrocellulose membrane flows through a quality control C line along with the test sample to be detected;
when a sample to be detected is dripped on the sample pad, if a carcinogen AFB1 exists in the sample to be detected, a carcinogen AFB1 is combined with AFB1 antibodies marked on UCNPs to form a compound, the compound flows into the nitrocellulose membrane 5 together, the compound fixed on the UCNPs reacts with AFB1 antigens fixed on a detection T line, part of the compound is fixed on the detection T line, the rest part of the compound flows through a quality control C line along with the sample to be detected, and the carcinogen AFB1 fixed on the UCNPs is combined with goat anti-mouse IgG antibodies fixed on the quality control C line and is fixed on the quality control C line.
(3) When detecting single component substances, 980nm laser is used for excitation to obtain detection signals of a T line and a C line, 808nm laser is used for excitation to obtain detection signals of the T line and the C line, and the detection signals of the latter wave band are used for authenticating the detection signals of the former wave band.
The chromatography test strip for the double-excitation double-emission upconversion nanoparticles and the detection method thereof also comprise the following steps:
(1) treatment of bond pads
Taking a glass cellulose membrane 5 as a bonding pad material, firstly soaking the bonding pad 3 in a treatment solution of the bonding pad 3 for 24 hours, drying at 37 ℃, dispersing a solution of double-excitation double-emission up-conversion nanoparticles marked with AFB1 antibody to be detected in a PBS (phosphate buffer solution) containing 0.05-10% of BAS (basic organic solvent), 0.1-2% of Tween-20, 0.27M NaCl and 0.1M Hepes, wherein 0.01M, pH is 7.0-8.0, then placing the bonding pad 3 in the solution of the double-excitation double-emission up-conversion nanoparticles marked with AFB1 antibody to be detected obtained in the step 2.1, soaking for 4 hours, and drying at 37 ℃ to finish the treatment of the bonding pad; the pad treatment solution was a PBS buffer solution containing 0.01M, pH ═ 7 with a mass concentration of 1% BAS and 2% Tween-20.
(2) Treatment of nitrocellulose membranes
The nitrocellulose membrane was divided into two parts:
streaking AFB1 antigen to be detected on the first portion at a parameter of 1.3 μ L/cm to form a detection T line coating AFB1 antigen;
drawing lines on the second part of the goat anti-mouse IgG antibody according to the parameter of 1.5 mu L/cm to form a quality control C line; drying to finish the treatment of the nitrocellulose membrane;
(3) assembly of test strips
The sample pad 2 after the adhesive treatment, the bonding pad 3 after the adhesive treatment, the nitrocellulose membrane 5 after the adhesive treatment, and finally the absorbent paper 4 are adhered in sequence from the front end to the rear end of the adhesive plastic base plate 1.
The preparation steps before detection are detailed as follows: and dropwise adding solutions of detection substances with different concentrations on the sample pad 2 for detection, respectively exciting by 980nm laser and 808nm laser after detection, and observing color changes of the detection region and the quality detection region. At the moment, after being excited by 980nm laser, the detection T line can emit red light, and the fluorescence intensity is reduced along with the increase of the concentration of the aflatoxin; the quality control C line also emits red light, and the fluorescence intensity is kept unchanged. Then, a T line obtained after 808nm laser excitation emits green light, and the fluorescence intensity is reduced along with the increase of the concentration of the aflatoxin; the quality control C line also emits green light, the fluorescence intensity is kept unchanged, and a contrast curve of the fluorescence intensity of the T line and the fluorescence intensity of the C line is established through the fluorescence intensity obtained by excitation of two near infrared lights. And comparing the detection signals of the two wave bands to realize a detection means of double authentication.
And preferably, a T-line fluorescence intensity ratio curve and a C-line fluorescence intensity ratio curve are established, and the quantitative detection of the components contained in the sample to be detected is realized by comparing the T-line fluorescence intensity ratio curve with the standard curve.
FIG. 2 is a schematic diagram of the reaction for detecting the immunoreaction of a single component.
Example 2:
referring to fig. 3, the embodiment of the present invention provides a preparation method, particles, a test strip and a detection method for preparing dual-excitation dual-emission particles that excite red light at 980nm and green light at 808nm, so as to implement simultaneous detection of AFB1 and AFB2 (two types in AFT), the specific steps are substantially the same as those in embodiment 1, and the difference is that:
1. preparation of UCNPs
1.1, preparing double-excitation double-emission up-conversion nanoparticles with a three-layer core-shell structure by adopting a seed crystal method, wherein NaYF doped with Yb, Tm and Er is used4The nano-particles are cores, and Yb doped NaYF is wrapped outside the cores4A first shell layer, andyb and Nd doped NaYF is wrapped outside the first shell layer4A second shell layer which forms Yb, Tm, Er and Nd co-doped NaYF4:Yb/Tm/Er@NaYF4:Yb@NaYF4Yb/Nd core-shell structure; the up-conversion 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 comprises the following specific steps:
a. synthesis of NaYF doped with Yb, Tm and Er4Nanoparticles as inner core
10mL of Oleic Acid (OA), 10mL of Octadecene (ODE), 0.84g of NaF solid, and Yb (OAc) were weighed out30.0683g、Tm(OAc)30.0017g、Er(OAc)30.2752g, adding into a three-neck flask A, magnetically stirring, heating to 110-120 ℃, keeping for 10min, and then vacuumizing to remove water and oxygen; removing all and then introducing N2Heating to 300 ℃ and reacting for 1 h;
b. wrapping Yb doped NaYF outside the inner core4First shell
4mL of Oleic Acid (OA), 4mL of Octadecene (ODE), 0.4788g of NaF solid, and Yb (OAc) were weighed out30.07g 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 N2Heating 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)30.0525g、Nd(OAc)30.4333g, 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 N2After 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 ℃; cooling to room temperature after the reaction is finished, putting the reaction liquid in the three-neck flask A into a centrifugal tube, and centrifugally separating to obtain the obtained nano particles;
d. stripping oleic acid from the surface of the nanoparticles obtained in step c
And c, taking the nano particles obtained in the step c, adding a mixed solution of ethanol with the pH value of 1 and concentrated hydrochloric acid (7.5mL of ethanol and 62.5 mu L of concentrated hydrochloric acid), carrying out ultrasonic dispersion uniformly, then carrying out vibration while carrying out ultrasonic treatment for 30min, then centrifuging, removing a supernatant, adding a mixed solution of ethanol with the pH value of 4 and concentrated hydrochloric acid (7.5mL of ethanol and 7.5mL of concentrated hydrochloric acid), carrying out ultrasonic dispersion uniformly, carrying out vibration while carrying out ultrasonic treatment for 30min, then carrying out centrifugal separation again, and washing the obtained nano particles with water to obtain the water-soluble double-excitation double-emission up-conversion nano particles.
The up-conversion nanoparticles prepared by the method can simultaneously detect substances containing AFB1 and AFB2, and can also realize double authentication on a detected substance of a single component of AFB1 or AFB2 to be detected; the up-conversion nano particles can emit any two wave bands of red light, green light or blue light under the excitation of two different near infrared light.
2. Preparation of UCNPs-antibodies
2.1 coating a layer of polyethylene-propylene copolymer on the surface of the upconversion nanoparticles by electrostatic adsorption
Taking 5mg of the double-excitation double-emission up-conversion nanoparticles obtained in the step 1.1, taking the ratio of the mass of the up-conversion nanoparticles to the mass of the polypropylene solution as 4:1, dispersing the up-conversion nanoparticles in 1mg/mL of polypropylene buffer solution, carrying out shaking table reaction at normal temperature for 12-14 h, then carrying out centrifugal separation at 4 ℃ to obtain the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene, dispersing the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene in PBS buffer solution containing 0.01M, pH-7.0-8.0, and storing at 4 ℃, thus completing the preparation of the solution of the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene.
2.2, marking the AFB1 and AFB2 antibodies on the surface of the double-excitation double-emission upconversion nano particle wrapping the polypropylene through electrostatic adsorption and the like
Then adding EDC and NHS into the solution of the double-excitation double-emission upconversion nanoparticles wrapped with the polypropylene according to the carboxyl ratio of the upconversion nanoparticles to EDC of 1:10 and the ratio of EDC to NHS of about 1:5, carrying out shaking table reaction for 30min at normal temperature, and then carrying out centrifugal separation at 4 ℃ to activate the particles. Adding the antibodies of AFB1 and AFB2 to be detected into a solution of double-excitation double-emission upconversion nanoparticles wrapping polypropylene according to the mass ratio of the mass of the upconversion nanoparticles to the antibody of the carcinogen to be detected of 100-300: 1, carrying out shaking table reaction for 4 hours at normal temperature, carrying out centrifugal separation at 4 ℃ to obtain double-excitation double-emission upconversion nanoparticles marking the antibodies of AFB1 and AFB2 to be detected, and carrying out storage at 4 ℃ in PBS (phosphate buffered saline) buffer solution containing 50mM glycine and 0.1-3% Tween-20 and 0.01M, pH-7.0-8.0, so as to finish the preparation of the solution of the double-excitation double-emission upconversion nanoparticles marking the antibodies of AFB1 and AFB2 to be detected.
3. The preparation of the chromatography test strip specifically comprises the following steps:
the test strip of this embodiment includes a bottom plate 1, a sample pad 2, a combination pad 3, a nitrocellulose membrane 5, and a water absorbent paper 4. The double-excitation double-emission up-conversion nanoparticles are fixed on the binding pad 3, and antibodies of AFB1 and AFB2 to be detected are marked on the up-conversion nanoparticles; two detection T lines are arranged in different areas on the nitrocellulose membrane 5, wherein T1 is coated with AFB1 antigen, and T2 is coated with AFB2 antigen; and a quality control C line is arranged at the same time, and a goat anti-mouse IgG antibody is coated on the quality control C line.
(1) Treatment of the bonding pad 3
Taking a glass cellulose membrane 5 as a material of a bonding pad 3, firstly soaking the bonding pad in a treatment solution of the bonding pad 3 for 24 hours, drying at 37 ℃, dispersing a solution of double-excitation double-emission up-conversion nanoparticles for marking AFB1 and AFB2 antibodies to be detected in a PBS (phosphate buffer solution) containing 0.05-10% of BAS (basic organic solvent), 0.1-2% of Tween-20, 0.27M NaCl and 0.1M Hepes, wherein 0.01M, pH is 7.0-8.0 by mass concentration, then placing the bonding pad in the solution of the double-excitation double-emission up-conversion nanoparticles for marking AFB1 and AFB2 antibodies to be detected obtained in the step 2.1, soaking for 4 hours, and drying at 37 ℃, thus finishing the treatment of the bonding pad 3; the treatment solution for the conjugate pad 3 was a PBS buffer solution containing 0.01M, pH ═ 7 with a mass concentration of 1% BAS and 2% Tween-20.
(2) Treatment of nitrocellulose Membrane 5
The nitrocellulose membrane 5 was divided into two parts:
marking two carcinogens AFB1 antigen and AFB2 antigen to be detected in different areas of the first part according to the parameter of 1.3 mu L/cm, and respectively forming a detection T1 line and a detection T2 line of the antibody coated with the corresponding carcinogens;
drawing lines on the second part of the goat anti-mouse IgG antibody according to the parameter of 1.5 mu L/cm to form a quality control C line; drying to finish the treatment of the nitrocellulose membrane;
(3) assembly of test strips
The sample pad 2 after the adhesive treatment, the bonding pad 3 after the adhesive treatment, the nitrocellulose membrane 5 after the adhesive treatment, and finally the absorbent paper 4 are adhered in sequence from the front end to the rear end of the adhesive plastic base plate 1.
4. Detection method and process of up-conversion test strip
During detection, a sample to be detected is dripped on the sample pad 1, if carcinogens AFB1 and AFB2 exist in the sample to be detected, the carcinogens AFB1 and AFB2 are combined with AFB1 and AFB2 antibodies marked on UCNPs to form a compound, the compound flows into the nitrocellulose membrane 5 together, the compound fixed on the UCNPs respectively reacts with AFB2 antigens fixed on AFB1 and T2 lines fixed on a detection zone T1 line, parts of the compound are respectively fixed on the detection T1 and T2 lines, the rest of the compound flows through a quality control C line along with the sample to be detected, and the compound fixed on the UCNPs is combined with a goat anti-mouse IgG antibody fixed on the quality control C line and is fixed on the quality control C line.
At this time, the detection T1 line-immobilized upconverting nanoparticles exhibited red luminescence under excitation by 980nm excitation light, with the fluorescence intensity decreasing with increasing AFB1 concentration. The upconversion nanoparticles fixed on the quality control C line display red light and the fluorescence intensity remains unchanged; detection of T2 wire-immobilized upconverted nanoparticles under excitation with 808nm excitation light exhibited green luminescence with decreasing fluorescence intensity with increasing concentration of AFB 2. The upconversion nanoparticles immobilized on the quality control C line showed green light with the fluorescence intensity remaining unchanged. The qualitative detection for simultaneously detecting the two components contained in the sample to be detected can be realized by the difference of detection signals of AFB1 and AFB 2. FIG. 3 is a reaction scheme for simultaneously detecting two component immune responses.
In the embodiment, when the two components are detected simultaneously, detection signals of two different T lines and C lines are obtained by respectively exciting the two components by 980nm laser and 808nm laser, and one component in a sample to be detected is judged according to the difference of the detection signals, so that qualitative detection for simultaneously detecting the two components contained in the sample to be detected is realized.
In addition, a T-line fluorescence intensity ratio curve and a C-line fluorescence intensity ratio curve can be established in advance, and the quantitative detection of the components contained in the sample to be detected can be realized through comparison with a standard curve.
The above description is only exemplary of the present invention and should not be taken as limiting the invention, and any modifications, equivalents, improvements and the like that are made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. A preparation method of double-excitation double-emission up-conversion nanoparticles is characterized by comprising the following steps:
(1) the method adopts a seed crystal method to prepare double-excitation double-emission up-conversion nanoparticles with a three-layer core-shell structure, and NaYF doped with Yb, Tm and Er4The nano-particles are cores, and Yb doped NaYF is wrapped outside the cores4A first shell layer, and Yb and Nd doped NaYF is wrapped outside the first shell layer4A second shell layer which forms Yb, Tm, Er and Nd co-doped NaYF4:Yb/Tm/Er@NaYF4:Yb@NaYF4Yb/Nd core-shell structure; the water-soluble dual-excitation dual-emission up-conversion nano-particle UCNPs are prepared, and the up-conversion nano-particle emits 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 dual-emission upconversion nanoparticles (UCNPs) according to claim 1, wherein the step (1) specifically comprises the following steps:
a. synthesis of NaYF doped with Yb, Tm and Er4The nano-particles are used as the inner core,
10mL of Oleic Acid (OA) and 10m of Octadecene (ODE) were weighedL, NaF solid 0.84g, Yb (OAc)30.0683g、Tm(OAc)30.0017g、Er(OAc)30.2752g, adding into a three-neck flask A, magnetically stirring, heating to 110-120 ℃, keeping for 10min, and then vacuumizing to remove water and oxygen; removing all and then introducing N2Heating to 300 ℃, and reacting for 1 h;
b. wrapping Yb doped NaYF outside the inner core4First shell
4mL of Oleic Acid (OA), 4mL of Octadecene (ODE), 0.4788g of NaF solid, and Yb (OAc) were weighed out30.07g of the mixture is added into a three-neck flask B, the mixture is magnetically stirred and heated to 110 to 120 ℃, the temperature is kept for 10min, and then the mixture is vacuumized to remove water and oxygen; removing all and then introducing N2Heating to 150 ℃ and 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 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)30.0525g、Nd(OAc)30.4333g, 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 N2After 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 ℃; cooling to room temperature after the reaction is finished, putting the reaction liquid in the three-neck flask A into a centrifugal tube, and centrifugally separating to obtain the obtained nano particles;
d. stripping oleic acid from the surface of the nanoparticles obtained in step c
And c, taking the nano-particles obtained in the step c, adding a mixed solution of ethanol with the pH value of 1 and concentrated hydrochloric acid (7.5mL of ethanol and 62.5 mu L of concentrated hydrochloric acid), carrying out ultrasonic dispersion uniformly, then carrying out vibration while carrying out ultrasonic treatment for 30min, then centrifuging, removing a supernatant, adding a mixed solution of ethanol with the pH value of 4 and concentrated hydrochloric acid (7.5mL of ethanol and 7.5mL of concentrated hydrochloric acid), carrying out ultrasonic dispersion uniformly, carrying out vibration while carrying out ultrasonic treatment for 30min, then carrying out centrifugal separation again, and washing the obtained nano-particles with water to obtain the water-soluble dual-excitation dual-emission upconversion nano-particle UCNPs.
3. The method for preparing dual-excitation dual-emission upconversion nanoparticles (UCNPs) according to claim 1, further comprising the steps of:
(2) surface treatment of the upconversion nanoparticles: the method is characterized in that a layer of polypropylene is coated on the surface of the upconversion nanoparticles through the actions of electrostatic adsorption and the like, and specifically comprises the following steps:
taking 5mg of the double-excitation double-emission up-conversion nanoparticles prepared in the step (1), taking the ratio of the mass of the up-conversion nanoparticles to the mass of the polypropylene solution as 4:1, dispersing the up-conversion nanoparticles in 1mg/mL of polypropylene buffer solution, carrying out shaking table reaction at normal temperature for 12-14 h, then carrying out centrifugal separation at 4 ℃ to obtain the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene, dispersing the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene in PBS buffer solution containing 0.01M, pH-7.0-8.0, and storing at 4 ℃, thus completing the preparation of the solution of the double-excitation double-emission up-conversion nanoparticles coated with the polypropylene.
4. The method for preparing dual-excitation dual-emission upconversion nanoparticles (UCNPs) according to claim 1, further comprising the steps of:
(3) preparation of UCNPs-antibodies: the surface of the double-excitation double-emission up-conversion nano-particles wrapped by the polypropylene is marked with the antibody of AFB1 and AFB2 or the compound antibody thereof through the action of electrostatic adsorption and the like, so that the double-excitation double-emission up-conversion nano-particles marked with the antibody of AFB1 and AFB2 to be detected or the compound antibody thereof are obtained.
5. The double-excitation double-emission upconversion nanoparticle prepared by the method according to any one of claims 1 to 4, wherein the upconversion nanoparticle can simultaneously detect a substance containing two components of AFB1 and AFB2, and can also realize double certification for a detected substance containing a single component of AFB1 or AFB2 to be detected; the up-conversion nano particles can emit any two wave bands of red light, green light or blue light under the excitation of two different near infrared light.
6. A chromatography test strip adopting the double-excitation double-emission up-conversion nanoparticles as claimed in claim 5 is characterized by comprising a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially arranged on the bottom plate from left to right; wherein the nitrocellulose membrane is arranged in the middle of the bottom plate, the sample pad is arranged on the left side of the nitrocellulose membrane, and a combination pad is arranged between the nitrocellulose membrane and the sample pad; the absorbent paper is arranged on the right side of the nitrocellulose membrane; wherein, the sample pad and the combination pad, the combination pad and the nitrocellulose membrane, and the nitrocellulose membrane and the absorbent paper are partially laminated with each other; the binding pad is provided with double-excitation double-emission up-conversion nanoparticles, and antibodies of AFB1 and/or AFB2 to be detected are/is marked on the up-conversion nanoparticles; one or two detection T lines are arranged in different areas on the nitrocellulose membrane, wherein T1 is coated with AFB1 antigen, and T2 is coated with AFB2 antigen; and a quality control C line is arranged at the same time, and a goat anti-mouse IgG antibody is coated on the quality control C line.
7. The chromatography test strip of double-excitation double-emission up-conversion nanoparticles as claimed in claim 6, wherein the substrate is a viscous plastic substrate, and a viscous substance is arranged on the upper surface of the substrate; the assembly sequence is as follows: and sequentially adhering the treated sample pad, the bonded combination pad, the bonded nitrocellulose membrane and the absorbent paper from the left side to the right side of the adhesive plastic base plate.
8. The dual-excitation dual-emission chromatography test strip for up-conversion nanoparticles as claimed in claim 6, wherein: the length of the viscous plastic bottom plate is 60 mm; the length of the sample pad, the length of the combination pad, the length of the nitrocellulose membrane and the length of the absorbent paper are 8-17 mm, and contact areas of all parts are mutually overlapped and pressed for 1-3 mm during assembly.
9. The detection method of the chromatography test strip of the double-excitation double-emission up-conversion nanoparticles according to any one of claims 6 to 8, wherein the qualitative detection of the detection object contained in the sample to be detected is realized according to the color change of the up-conversion nanoparticles in the test strip detection area, and the method specifically comprises the following steps:
(1) dropwise adding a test sample to be detected on the sample pad, wherein the test sample to be detected can be combined with the corresponding antibody of the substance to be detected marked on the up-conversion nanoparticles, and flows into the nitrocellulose membrane together, and part of the nitrocellulose membrane is fixed on the T line to be detected, and the rest of the nitrocellulose membrane flows through the quality control C line along with the test sample to be detected;
(2) when detecting a single-component substance, firstly obtaining detection signals of a T line and a C line after being excited by 980nm laser, then obtaining detection signals of the T line and the C line after being excited by 808nm laser, and authenticating the detection signals of the former wave band by using the detection signals of the latter wave band;
(3) when the two components are detected simultaneously, detection signals of two different T lines and C lines are obtained by respectively exciting the two components by 980nm laser and 808nm laser, and one component in a sample to be detected is judged according to the difference of the detection signals, so that qualitative detection for simultaneously detecting the two components in the sample to be detected is realized.
10. The method for detecting a chromatography test strip of a double-excitation double-emission up-conversion nanoparticle according to claim 9, wherein the step (1) further comprises the following steps:
(11) before detection, solutions of detection objects with different concentrations are respectively dripped on a sample pad for detection; and (3) obtaining the fluorescence intensity of the corresponding detection T line and the fluorescence intensity of the corresponding quality detection C line through laser excitation of 980nm and 808nm, 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 quantitative detection of the components contained in the sample to be detected.
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