CN108918863B

CN108918863B - Preparation method of up-conversion aptamer test strip for rapid detection of ochratoxin A

Info

Publication number: CN108918863B
Application number: CN201810502981.8A
Authority: CN
Inventors: 吴世嘉; 刘丽红; 段诺; 王周平; 虞倩茹
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-05-23
Filing date: 2018-05-23
Publication date: 2021-05-11
Anticipated expiration: 2038-05-23
Also published as: CN108918863A

Abstract

The invention discloses a preparation method of an up-conversion aptamer test strip for rapidly detecting ochratoxin A. It is characterized in that: the method comprises the following steps of modification of an up-conversion luminescent nano material: will contain Yb³⁺、Y³⁺、Er³⁺Adding oleic acid and octadecylene into the inorganic salt, mixing, stirring, introducing inert gas, and heating to form a uniform solution; dissolving ammonium fluoride and sodium hydroxide in methanol, adding the solution into the methanol, heating and evaporating to remove the methanol, introducing inert gas for reaction, adding a condensation reflux device, and heating for reaction; mixing polyacrylic acid and ethanol, dispersing the up-conversion luminescent nano material in a chloroform solution, adding the solution into the mixed solution of polyacrylic acid and ethanol, and stirring for reaction to obtain a modified up-conversion luminescent nano material; an upconverter aptamer probe was prepared. The invention provides a novel chromatography test strip technology, which can effectively improve the sensitivity of a test strip, reduce the manufacturing cost and achieve the aim of quantitative detection.

Description

Preparation method of up-conversion aptamer test strip for rapid detection of ochratoxin A

Technical Field

The invention belongs to the technical field of mycotoxin detection, and particularly relates to a preparation method of an up-conversion aptamer test strip for rapid detection of ochratoxin A.

Background

Ochratoxins are another mycotoxin which has attracted widespread worldwide attention following aflatoxins. It is an important group of food-contaminating mycotoxins produced by 7 aspergillus species in the genus aspergillus and 6 penicillium species in the genus penicillium, 4 of which ochratoxin a is the most toxic, most widespread, most toxic in production, most polluting in agricultural products, and most closely related to human health. Chinese patent 201611111887.7, a potato virus rapid detection test strip, a preparation method and applications thereof, the test strip uses an antibody as a recognition substance to achieve the purpose of target detection. However, the preparation period of the antibody is long, the process is complex, the preparation of the test strip is time-consuming and labor-consuming, and only qualitative detection can be realized. Chinese patent 201710641207.0 discloses a colloidal gold chromatography rapid diagnostic test strip for detecting acetylcholinesterase and a preparation method thereof, wherein the technique uses colloidal gold as a marker and an antibody as an identifier, which can realize rapid detection of acetylcholinesterase but cannot realize the purpose of quantification. And the application of the antibody increases the preparation difficulty and cost of the antibody. Chinese patent 201510662526.0 discloses an immunofluorescence test strip for quantitatively detecting swine foot-and-mouth disease virus, which is used together with a fluorescence detector to achieve the purpose of quantitative detection. However, the adopted recognition substance is still an antibody, so that the preparation difficulty and the cost of the antibody are increased.

Therefore, the preparation of a test paper with high speed, accuracy, sensitivity and specificity is a technical problem to be solved in the prior art.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made in view of the above-mentioned technical drawbacks.

Therefore, in one aspect of the present invention, the present invention overcomes the defects in the prior art, and provides a preparation method of an upconverter aptamer test strip for rapid detection of ochratoxin a.

In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of an up-conversion aptamer test strip for rapid detection of ochratoxin A is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

preparing and modifying an up-conversion luminescent nano material: will contain Yb³⁺、Y³⁺、Er³⁺Adding oleic acid and octadecylene into the inorganic salt, mixing, stirring, introducing inert gas, and heating to form a uniform solution; dissolving ammonium fluoride and sodium hydroxide in methanol, adding the solution, heating and evaporating to remove the methanol, introducing inert gas for reaction, adding a condensation reflux device, heating for reaction, and obtaining the up-conversion luminescent nano material; mixing polyacrylic acid and ethanol, and dispersing the up-conversion luminescent nano material in chlorineAdding the imitation solution into a polyacrylic acid and ethanol mixed solution, and stirring for reaction to obtain a modified up-conversion luminescent nano material;

preparation of upconverter aptamer probes: coupling the modified up-conversion luminescent nano material with an aptamer to prepare an up-conversion aptamer probe;

immobilization of aptamer on test strip nitrocellulose membrane: DNA complementary to the recognition sequence and non-recognition sequence of the aptamer_OTAAnd DNA_{poly A}And the streptavidin is fixed on a detection line and a quality control line of the nitrocellulose membrane.

As a preferred scheme of the preparation method of the test strip of the up-conversion aptamer for rapid detection of ochratoxin A, the test strip comprises the following steps: of the up-conversion luminescent nanomaterial, YCl₃78% of YbCl₃20% of ErCl₃The content is 2%, and the content of the oleic acid is as follows by volume ratio: octadecene 2: 5.

as a preferred scheme of the preparation method of the test strip of the up-conversion aptamer for rapid detection of ochratoxin A, the test strip comprises the following steps: the up-conversion luminescent nano material is modified by including YbCl₃·6H₂O、YCl₃·6H₂O and ErCl₃Adding 6mL of oleic acid and 15mL of octadecene, gradually heating to 160 ℃ under the environment of stirring and introducing nitrogen to enable the reagent to form a uniform solution, naturally cooling to room temperature, and weighing 4mmol of NH₄F and 2.5mmol of NaOH are dissolved in 10mL of methanol solution, the solution is dropwise added into a three-neck flask, the temperature is raised to 60 ℃, magnetic stirring is carried out for 30min, and then the temperature is gradually raised to evaporate and remove the methanol; and (3) continuously introducing argon, adding a condensation reflux device, heating to 300 ℃, continuously reacting for 1h, closing the device after the reaction is finished, naturally cooling to room temperature, centrifugally cleaning the material with water and ethanol for three times, and drying at 70 ℃ for 8h to obtain the up-conversion luminescent nano material.

As a preferred scheme of the preparation method of the test strip of the up-conversion aptamer for rapid detection of ochratoxin A, the test strip comprises the following steps: the method comprises the following steps of modifying the up-conversion luminescent nano material, uniformly stirring 200mg of polyacrylic acid and 8mL of absolute ethyl alcohol solution, dispersing 30mg of the up-conversion luminescent nano material in 4mL of chloroform solution, dropwise adding the solution into the polyacrylic acid and the absolute ethyl alcohol solution, reacting for 24 hours under magnetic stirring, and centrifugally collecting the material to obtain the modified up-conversion luminescent nano material.

As a preferred scheme of the preparation method of the test strip of the up-conversion aptamer for rapid detection of ochratoxin A, the test strip comprises the following steps: the preparation of the up-conversion aptamer probe comprises the steps of dispersing 20mg of the modified up-conversion luminescent nano material in 4mL of 10mM morpholine ethanesulfonic acid buffer solution, carrying out ultrasonic dispersion, adding 0.2mL of 5mg/mL N-hydroxy thiosuccinimide and 0.1mL of 5mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution, oscillating for 2 hours in a shaker at 37 ℃ for carboxyl activation, dispersing in 2mL of aqueous solution after centrifugal washing, adding an amination aptamer, incubating for 24 hours at 37 ℃, and dispersing in 2mL of water after centrifugal washing to obtain the up-conversion aptamer probe.

As a preferred scheme of the preparation method of the test strip of the up-conversion aptamer for rapid detection of ochratoxin A, the test strip comprises the following steps: the aptamer is fixed on the nitrocellulose membrane of the test strip, and the method comprises the steps of taking 100 mu L of 0.125mg/mL streptavidin and respectively adding 100 mu M DNA_OTAAnd DNA_{poly A}Shaking and mixing uniformly, placing the mixed solution in a shaking table at 37 ℃ for incubation for 30min, respectively spraying the two mixed solutions on a nitrocellulose membrane by using a dot crystal membrane spraying instrument, wherein the spraying amount is 0.8 mu L/mL, the distance between two lines is 0.5mm, and placing the treated nitrocellulose membrane in an oven at 37 ℃ for 1 h.

As a preferred scheme of the preparation method of the test strip of the up-conversion aptamer for rapid detection of ochratoxin A, the test strip comprises the following steps: the nitrocellulose membrane comprises a SartoriusCN140 nitrocellulose membrane.

As a preferred scheme of the preparation method of the test strip of the up-conversion aptamer for rapid detection of ochratoxin A, the test strip comprises the following steps: the aptamer sequence Apt_{OTA 1}Comprises the following steps: 5' -NH₂-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGATCGGGTGTGGGTG GCGTAAAG GGAGCATCGGACA-3'; the aptamerComplementary sequence DNA of recognition sequence and non-recognition sequence_OTAThe sequence of the DNA is 5' -bio-TGTCCGATGCTCCCTTTACGCCACCCACACCCG ATC-3_polyAThe sequence is 5 '-bio-AAAAAAAAAAAAAA AAAAAAAAAAAAAAAA-3'.

The invention has the beneficial effects that: the invention provides a novel chromatography test strip technology, which can effectively improve the sensitivity of a test strip, reduce the manufacturing cost and achieve the aim of quantitative detection. The aptamer is applied to the chromatographic test strip and is used as an identification molecule to replace an antibody for target identification. Meanwhile, the upconversion luminescent nano material is used as a signal marker of the test strip, so that the excellent optical performance of the upconversion material is fully utilized, the sensitivity of the test strip is improved, the background interference is reduced, the result is more accurate, and the aim of accurate quantification is fulfilled.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 shows three upconversion luminescent materials UCNPs of the present invention₁、UCNPs₂、UCNPs₃A TEM image of (a).

FIG. 2 is a graph of luminescence spectra of three upconversion luminescent materials UCNPs of the present invention.

FIG. 3 is a graph showing the results of three UCNPs of the present invention added to a test strip.

FIG. 4 is a graph showing the results of three up-conversion probes of the present invention added dropwise to test strips with different nitrocellulose membranes.

FIG. 5 is a chart showing the test results of OTA test paper strip with loading amount of 10 μ L (a), 20 μ L (b), 30 μ L (c) and 40 μ L (d), respectively.

FIG. 6 is a process flow diagram of the present invention.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The invention modifies up-conversion luminescent nano materials (UCNPs) by polyacrylic acid, so that the surfaces of the UCNPs have carboxyl groups and are connected with amino-modified aptamers (Apt)_OTA1) Formation of Up-converting aptamer Probe UCNPs-Apt_OTA1. DNA complementary to the recognition sequence and non-recognition sequence of the aptamer_OTAAnd DNA_{poly A}And the streptavidin is fixed on a detection line (T line) and a quality control line (C line) of the nitrocellulose membrane. UCNPs-Apt when ochratoxin A (OTA) does not exist in the solution to be detected_OTAThe fluorescent probe is captured by complementary sequences on a T line and a C line respectively, and two green luminous strips can be observed under a 980nm laser emitter; when OTA exists in the liquid to be detected, UCNPs-Apt_OTA1The target is captured to form a composite structure. The recognition sequence was occupied and could not bind to the complementary sequence of the T-line DNAOTA, thus detecting the reduction or disappearance of line banding at the 980nm laser emitter. But the complementary sequence DNA on the C-line, whether or not the target is present_polyACan capture UCNPs-Apt by base complementary pairing_OTA1。

Example 1:

synthesis of upconversion luminescent nanomaterials UCNPs₁: weighing YbCl₃·6H₂O、YCl₃·6H₂O and ErCl₃(78％Y³⁺，20％Yb³⁺，2％Er³⁺) Into a 100mL three-necked flask, 6mL oleic acid and 15mL octadecene were added. Under the environment of stirring and introducing nitrogen, the temperature is gradually increased to 160 ℃ to enable the reagent to form a uniform solution, and the solution is naturally cooled to the room temperature. Weighing 4mmol of NH₄F and 2.5mmol of NaOH are dissolved in 10mL of methanol solution. Dropwise adding the solution into a three-neck flask, heating to 60 ℃, magnetically stirring for 30min, gradually heating, and evaporating to remove methanol. Continuously introducing argon, adding a condensing reflux device, heating to 300 ℃, and continuously reacting for 1 h. After the reaction is finished, the device is closed, and the reaction product is naturally cooled to room temperature. The material was washed three times with water and ethanol and dried at 70 ℃ for 8h to give a white solid powder which was stored for use.

Polyacrylic acid is selected as a ligand exchanger, and carboxylation modification is carried out at normal temperature: a round-bottomed flask was charged with 200mgPAA and 8mL of an absolute ethanol solution, and the mixture was stirred well to dissolve it sufficiently. 30mg of oleic acid modified NaYF4 Yb, Er upconverter was weighed out and dispersed in 4mL of chloroform solution and added dropwise to the round bottom flask. And after magnetic stirring for 24 hours, centrifugally collecting the material, centrifugally cleaning the material for three times by using ultrapure water to obtain the modified upconversion material modified by the carboxyl group, and storing the modified upconversion material for later use.

Preparation of upconverter aptamer probes: taking modified up-conversion luminescent materials UCNPs₁20mg of the complex was dispersed in 4mL of 10mM morpholine ethanesulfonic acid (MES) buffer solution, and after ultrasonic dispersion, 0.2mL of 5mg/mL N-hydroxythiosuccinimide (sulfo-NHS) and 0.1mL of 5mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution were added and the mixture was shaken in a shaker at 37 ℃ for 2 hours to activate the carboxyl groups. After centrifugation, UCNPs are washed₁Dispersing in 2mL of aqueous solution, adding an aminated aptamer Apt_OTA1(the final concentration of the aptamer is 1000nM), incubating at 37 ℃ for 24h, centrifuging and washing, and dispersing the material in 2mL of deionized water to obtain the up-conversion probe UCNPs coupled with the aptamer₁-Apt_OTA1。

And (3) taking 100 mu L of 0.125mg/mL streptavidin, respectively adding 100 mu M DNOTA and DNApoly A, shaking and uniformly mixing, and placing the mixed solution in a shaking table at 37 ℃ for incubation for 30 min. Using a point crystal film spraying instrument to spray twoThe seed mixed solution was sprayed onto sartorius cn140 nitrocellulose membranes at a spray rate of 0.8 μ L/mL with a line spacing of 0.5 mm. And (3) placing the treated nitrocellulose membrane in a 37 ℃ oven for 1h, taking out, placing in a sealing bag, and storing in a dry and dark environment for later use. And assembling and cutting the absorbent paper, the sample pad, the PVC base plate and the nitrocellulose membrane into test strips with the width of 4mm, and placing the test strips in a dry and light-proof environment for later use. FIG. 6 is a process flow diagram of the present invention. Aptamer sequence is Apt_{OTA 1}：5’-NH₂-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGATCGGGTGTGGGTGGCGTAAAG GGAGCATCGGACA-3’。

Detection of ochratoxin a in beer samples:

the OTA content in the beer sample is measured by the method. Pretreating a sample: and (3) placing the beer sample in a refrigerator at 4 ℃ for refrigeration for 30min, and carrying out ultrasonic treatment to obtain 40g of a degassed sample. Pouring the liquid into a 50mL volumetric flask, adding the extracting solution (150g of sodium chloride and 20g of sodium bicarbonate are added with water to a constant volume of 1L) to a constant volume, uniformly mixing, filtering by using glass fiber filter paper until the filtrate is clear and transparent, and collecting the filtrate. And then selecting OTA standard substances with different concentrations to be respectively added into the processed beer samples until the final concentration of the OTA is respectively 0ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 300ng/mL and 500 ng/mL. After mixing the mixed solution uniformly, 30 μ L of the mixed solution was added dropwise to a sample pad of a new ochratoxin A test strip, and after 1min, the mixed solution was washed with PBS (pH 7.4), and the results are observed and shown in Table 1.

The accuracy, sensitivity and the like of the constructed test strip are verified by adopting a standard addition recovery method, and the result shows that the recovery rate of ochratoxin A is 95.2-115.6%, and the on-site sensitive and rapid detection can be met.

Table 1 example 1 determination of OTA content in beer samples

Sample (I)	OTA concentration (ng/mL)	The result of the detection	Instrument test result (ng/mL)	Recovery rate
					1	0	Negative of	0	/
2	5	Positive for	5.21	104.25
					3	10	Positive for	10.51	105.10％
4	20	Positive for	23.11	115.56％
					5	50	Positive for	48.52	97.04％
6	100	Positive for	95.17	95.17％
					7	300	Positive for	/	/
8	500	Positive for	/	/

Example 2:

synthesis of upconversion luminescent nanomaterials UCNPs₂The specific method comprises the following steps: 0.3g NaOH, 1.5mL deionized water, 5mL oleic acid, and 10mL ethanol were added sequentially to a 100mL beaker and mixed with rapid stirring to form a clear homogeneous solution. Weigh 1.6mL Y (NO)₃)₃·6H₂O(0.5mol/L)，0.9mL Yb(NO₃)₃·5H₂O(0.2mol/L)，0.1mL Yb(NO₃)₃·5H₂O (0.2mol/L) was added dropwise to the above solution, and mixed with vigorous stirring. 0.168g of NaF was weighed out and dissolved in 4mL of deionized water, and added dropwise to the above mixed solution. After stirring vigorously for 15min, the mixture was transferred to a 100mL reaction vessel with a Teflon liner and allowed to react at 200 ℃ for 8 h. And after the reaction is finished, taking out the reaction kettle, naturally cooling to room temperature, taking the bottom precipitate, centrifugally washing with ethanol for three times, and drying at 70 ℃ for 8 hours to obtain white solid powder for storage and later use.

Polyacrylic acid (PAA) is selected as a ligand exchanger to replace an oleic acid ligand on the surface of the up-conversion material, and the temperature is highThe upconverting material is modified with carboxyl groups under heating so that it is soluble in polar solutions. In a three-necked flask, 0.5g of PAA was dissolved by adding to 10mL of diethylene glycol solution, heated to 110 ℃ and stirred vigorously for 15 min. Weighing 30mg of oleic acid modified NaYF₄: and dispersing the Yb and Er up-conversion materials into 2mL of toluene solution, quickly transferring the solution into the mixed solution, mixing, and stirring at a high temperature for 15min so as to evaporate toluene. Then the temperature is increased to 240 ℃ to react for 1 h. After the reaction is finished, stopping heating, cooling to room temperature, adding excessive dilute hydrochloric acid, and centrifuging to obtain a white precipitate. Centrifugally cleaning for three times by using ultrapure water to obtain the carboxyl group modified up-conversion material, and storing for later use.

Example 3:

synthesis of upconversion luminescent nanomaterials UCNPs₃The specific method comprises the following steps: take 0.18g Y₂O₃(28％)，0.788g Yb₂O₃(70%) and 0.022g Er₂O₃(2%) adding the above-mentioned mixture into nitric acid solution, heating to dissolve it and evaporating excess nitric acid to obtain the nitrate powder of the above-mentioned rare earth element. After dissolving in 8mL of deionized water, 2.1273g of ethylenediaminetetraacetic acid (EDTA) was added to adjust the pH of the solution to be weakly alkaline and sufficiently dissolve. 25mL of ethylene glycol solution was measured, and 0.4g of cetyltrimethylammonium bromide (CTAB) and the above-mentioned mixed solution were added thereto and stirred rapidly. 1.5mL of HF was added dropwise. After the solution is white milky colloid, 3.5mL of concentrated nitric acid is added, the mixture is uniformly stirred and then transferred to a 100mL reaction kettle with a polytetrafluoroethylene lining, and the reaction is carried out for 24 hours at the temperature of 195 ℃. And after the reaction is finished, closing the device, taking out the reaction kettle, and naturally cooling to room temperature. Taking the lower layer precipitate, adding hot water for washing, fully stirring and standing. After the solid precipitates, the upper liquid layer is discarded, hot water is added again for stirring, and the process is repeated for 3 times. And finally, adding an ethanol solution for ultrasonic dispersion, centrifuging to obtain the required material, drying for 8 hours at 70 ℃ to obtain solid powder, and storing for later use.

5mg of prepared UCNPs₃Dissolved in 2mL of cyclohexane solution, 0.1g of cetyltrimethylammonium bromide (CTAB) and 20mL of deionized water were added. The mixture was stirred vigorously at room temperature to evaporate excess cyclohexane and form a clear solution of UCNPs-CTAB. 10mL of the solution was transferred to 20mL of deionized water, and then added3mL of ethanol and 150. mu.L of a 2M NaOH solution were added, and stirring was continued. The temperature is raised to 70 ℃, 200 mu L of Tetraethoxysilane (TEOS) is added, and the reaction is carried out for 10 min. Centrifuging the material, and washing with alcohol three times to obtain silicon layer coated up-conversion material UCNPs₃@mSiO₂. Mixing newly prepared UCNPs₃@mSiO₂Transferred to 50mL of a solution containing 0.3g of NH₄NO₃In an ethanol solution at 60 ℃ for 2h to remove CTAB on the surface of the material. At this time, the upconverting material is soluble in a polar solution, but has no group capable of being linked to an aptamer on the surface, and thus, Aminopropyltriethoxysilane (APTES) is used as a silane coupling agent, and an amino group is linked to the surface of the upconverting material, and the upconverting material is centrifuged to obtain amino-coated UCNPs₃@mSiO₂A material.

Example 4:

up-conversion material and amination and carboxylation ochratoxin A aptamer Apt_{OTA 1}、Apt_{OTA 2}Coupling of (a):

in the invention, amino modified ochratoxin A aptamer sequence Apt_{OTA 1}：

5′-NH₂-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGATCGGGTGTGGGTGGCGTAAAG GGAGCATCGGACA-3'; carboxyl modified ochratoxin A aptamer sequence Apt_{OTA 2}：5′-COOH-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGATCGGGTGTGGGTGGCGTAAA GGGAGCATCGGACA-3′

Complementary sequence DNA of biotin-modified ochratoxin A aptamer recognition sequence_OTAComprises the following steps:

5′-bio-TGTCCGATGCTCCCTTTACGCCACCCACACCCGATC-3′，

complementary sequence DNA of biotin-modified ochratoxin A aptamer non-recognition sequence_polyAComprises the following steps:

5′-bio-AAAAAAAAAAAAAA AAAAAAAAAAAAAAAA-3′。

taking up-conversion materials UCNPs₁、UCNPs₂20mg each was dissolved in 4mL of 10mmol of morpholinoethanesulfonic acid (MES) buffer solution, and after ultrasonic dispersion, 0.2mL of 5mg/mL N-hydroxythiosuccinimide (sulfo-NHS) and 0.1mL of 5mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution were added to the solutionThe carboxyl group activation was performed by shaking for 2 hours in a shaker at 37 ℃. After centrifugation, UCNPs are washed₁、UCNPs₂Dispersing in 2mL of aqueous solution respectively, adding an aminated aptamer Apt_{OTA 1}；UCNPs₃Dispersing in 2mL of aqueous solution, adding a carboxylated aptamer Apt_{OTA 2}(the final concentration of the aptamer is 1000nM), incubating for 24h at 37 ℃, centrifuging and washing, and dissolving the material in 2mL deionized water to obtain three up-conversion probes UCNPs coupled with the aptamer₁-Apt_{OTA 1}、UCNPs₂-Apt_{OTA 1}And UCNPs₃-Apt_{OTA 2}。

Characterization of different upconversion luminescent nanomaterials:

for the prepared three NaYF₄: the Yb and Er up-conversion luminescence nano-material and the modified material thereof are characterized, and the results are shown in figure 1. FIG. 1a is a schematic representation of upconversion materials UCNPs₁A TEM image; FIG. 1b is a schematic representation of upconversion materials UCNPs₁Modified TEM image; FIG. 1c is a schematic representation of upconversion materials UCNPs₂A TEM image; FIG. 1d is a schematic representation of upconversion materials UCNPs₂Modified TEM image; FIG. 1e shows UCNPs as upconverting materials₃A TEM image; FIG. 1f is a schematic representation of upconversion materials UCNPs₃Modified TEM images.

TEM results show oleic acid coated NaYF₄: UCNPs (Yb, Er upconversion materials)₁The particle size is about 40 +/-6 nm (as shown in figure 1a), and the dispersion and the uniformity in a non-polar solution are better. The TEM characterization result of the modified material is shown in fig. 1b, and the result shows that the particle size of the modified upconverter material is about 40 ± 6nm, and the upconverter material has good dispersibility and uniformity.

To up-conversion luminescent nano material UCNPs₂Characterization was performed and the results are shown in FIG. 1 c. TEM results show oleic acid coated NaYF₄: the Yb and Er up-conversion material is square, the grain diameter is about 15 +/-8 nm, and the material has good dispersibility and uniformity in a non-polar solution. The TEM characterization result of the modified material is shown in FIG. 1d, and the result shows that the modified material is still square, the particle size is about 15 +/-8 nm, and the modified material has good dispersibility and uniformity.

For NaYF₄: UCNPs (carbon nano fibers) made of Yb and Er up-conversion luminescent nano materials₃Characterization was performed, the results are shown in FIG. 1e. TEM results show oleic acid coated NaYF₄: the Yb and Er up-conversion material is elliptical, has the grain diameter of about 40 +/-10 nm and has good uniformity in a non-polar solution. In order to make the material soluble in polar solution, the surface of the material is coated with a silicon layer, and then the silane-PEG-carboxyl is used to connect carboxyl around the material, as shown in FIG. 1 f. TEM results show that the surface of the material is successfully coated with the silicon layer, the material still has an oval shape, the particle size is about 50 +/-10 nm, the material has good uniformity, and the material is slightly aggregated.

Selection of up-conversion invention nano material:

the results of comparing the emission intensities of the three upconverters at the same concentration under 980nm laser irradiation are shown in FIG. 2. The UCNPs of the up-conversion material can be seen from the luminescence spectrogram₁And UCNPs₃The luminescence is better.

Besides the luminous intensity, the dispersibility of the material can influence the fluidity of the material on the test strip, and indirectly influence the performance of the test strip. If the material flowability is not good, the probe can be gathered on the sample pad and cannot pass through the nitrocellulose membrane, and the sensitivity of the test strip is seriously influenced.

Equal amounts of the three upconverter aptamer probes were added dropwise to the sample pad of the new OTA upconverter aptamer test strip and washed with PBS after 1 min. In order to observe the flow of the upconverting aptamer probe over the entire strip, the irradiation range needs to be enlarged. Therefore, a beam expander is connected to the 980nm laser transmitter to expand the beam and obtain a larger observation area, and as a result, fig. 3a shows UCNPs₁FIG. 3b represents UCNPs₂FIG. 3c represents UCNPs₃。

The experimental result shows that the UCNPs are the up-conversion materials₃Due to particle aggregation, the flowability is poor, a large number of probes remain on the sample pad, and T, C lines are all darker, so that the detection of an actual sample is not facilitated; UCNPs₂The fluidity is higher than that of UCNPs₃Good, but still some residue remained on the sample pad and the nitrocellulose membrane, and the T, C line was weak in brightness due to poor luminescence; and UCNPs₁The fluidity and the luminescence are better than those of the other two materials.

Selection of nitrocellulose membrane:

based on the above experiment, four different OTA up-conversion test strips were prepared from nitrocellulose membranes Whatman AE99, Millipore 135, Sartorius CN140 and Pall Vivid 170 with different pore sizes. New up-conversion aptamer probes UCNPs₁-Apt_{OTA 1}Drop-wise onto the sample pad and wash with PBS after 1 min. To observe probe migration throughout the strip, a beam expander was attached to the 980nm laser emitter to expand the range of observation, as shown in fig. 4, fig. 4a for Whatman AE99, fig. 4b for Millipore 135, fig. 4c for Sartorius CN140, and fig. 4d for Pall Vivid 170. As can be seen from the figure, the test strips made of two kinds of nitrocellulose membranes, Whatman AE99 and Millipore 135, have no residue after the up-conversion aptamer probe flows through, but the T line and the C line are light in color, which is not favorable for observing the change of the T line in the actual detection; the nitrocellulose membrane with the pore size of Pall Vivid 170 has the defects that the pore size is small, the flow rate is too slow, and the reaction time on the membrane is long, so that the colors of two strips are dark, but a large amount of residues exist on the membrane, background interference exists during quantification, and the accuracy of the result is influenced. The nitrocellulose membrane with the diameter of Sartorius CN140 is darker in color and almost has no residue, so the nitrocellulose membrane with the diameter of Sartorius CN140 is selected.

And (3) optimizing the sample loading amount:

the loading amount refers to the volume of the substrate and the up-conversion aptamer probe which are added to the test strip in a dropwise manner after incubation. Up-conversion aptamer probes with different volumes (10, 20, 30 and 40 mu L) are respectively mixed with OTA standard substances (the final concentration of OTA is 20ng/mL), and the mixture is added dropwise onto prepared test strips after incubation. After 1min, washing with PBS, observing the results, as shown in FIG. 5, the group A is blank control, the group B is actual detection result of 20ng/mL OTA, and the sample loading amounts are 10 μ L (a), 20 μ L (B), 30 μ L (c) and 40 μ L (d), respectively. As can be seen from the figure, when the amount of the sample is small, the OTA is added to produce a clear positive result due to the small number of the probes, but the color of the band is light, which is not favorable for observation. When the sample loading amount is large, the detection line can still be observed, and the sensitivity is poor. And a large amount of residues exist on the nitrocellulose membrane, background interference exists, and result quantification is difficult to achieve. Therefore, 30. mu.L is the optimum loading amount.

In conclusion, the invention provides a novel chromatography test strip technology, which can effectively improve the sensitivity of the test strip, reduce the manufacturing cost and achieve the purpose of quantitative detection, and the detection time of the invention only needs 1 min.

The aptamer is applied to the chromatographic test strip and is used as an identification molecule to replace an antibody for target identification. Meanwhile, the upconversion luminescent nano material is used as a signal marker of the test strip, so that the excellent optical performance of the upconversion material is fully utilized, the sensitivity of the test strip is improved, the background interference is reduced, the result is more accurate, and the aim of accurate quantification is fulfilled.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A preparation method of an up-conversion aptamer test strip for rapid detection of ochratoxin A is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

preparing and modifying an up-conversion luminescent nano material: will contain Yb³⁺、Y³⁺、Er³⁺Adding oleic acid and octadecylene into the inorganic salt, mixing, stirring, introducing inert gas, and heating to form a uniform solution; dissolving ammonium fluoride and sodium hydroxide in methanol, adding the solution, heating and evaporating to remove the methanol, introducing inert gas for reaction, adding a condensation reflux device, heating for reaction, and obtaining the up-conversion luminescent nano material; mixing polyacrylic acid and ethanol, dispersing the up-conversion luminescent nano material in a chloroform solution, adding the solution into the mixed solution of polyacrylic acid and ethanol, and stirring for reaction to obtain a modified up-conversion luminescent nano material;

fixing the aptamer complementary sequence on a test strip nitrocellulose membrane: DNA complementary to the recognition sequence and non-recognition sequence of the aptamer_OTAAnd DNA_polyAFixing the mixture on a detection line and a quality control line of the nitrocellulose membrane through streptavidin;

in the up-conversion luminescent nanomaterial, Y³⁺78% of Yb³⁺20% of Er³⁺The content is 2%, and the content of the oleic acid is as follows by volume ratio: octadecene 2: 5;

the up-conversion luminescent nano material is modified by including YbCl₃·6H₂O、YCl₃·6H₂O and ErCl₃Adding 6mL of oleic acid and 15mL of octadecene, gradually heating to 160 ℃ under the conditions of stirring and nitrogen introduction to enable the reagent to form a uniform solution, naturally cooling to room temperature, and weighing 4mmol of NH₄F and 2.5mmol NaOH are dissolved in 10mL of methanol solution, the solution is dropwise added into a three-neck flask, the temperature is raised to 60 ℃, magnetic stirring is carried out for 30min, and then the temperature is gradually raised to evaporate and remove the methanol; continuously introducing argon, adding the argon into a condensation reflux device, heating to 300 ℃, continuously reacting for 1h, closing the device after the reaction is finished, naturally cooling to room temperature, centrifugally cleaning the material with water and ethanol for three times, and drying at 70 ℃ for 8h to obtain the up-conversion luminescent nano material;

the aptamer is fixed on the nitrocellulose membrane of the test strip, and the method comprises the steps of taking 100 mu L of 0.125mg/mL streptavidin and respectively adding 100 mu M DNA_OTAAnd DNA_polyAShaking and uniformly mixing, placing the mixed solution in a shaking table at 37 ℃ for incubation for 30min, respectively spraying the two mixed solutions on a nitrocellulose membrane by using a dot crystal membrane spraying instrument, wherein the spraying amount is 0.8 mu L/mL, the distance between two lines is 0.5mm, and placing the treated nitrocellulose membrane in an oven at 37 ℃ for 1 h;

the nitrocellulose membrane comprises a SartoriusCN140 nitrocellulose membrane.

2. The method for preparing the test strip of upconverter aptamer for rapid detection of ochratoxin a of claim 1, which comprises: the method comprises the following steps of modifying the up-conversion luminescent nano material, uniformly stirring 200mg of polyacrylic acid and 8mL of absolute ethyl alcohol solution, dispersing 30mg of the up-conversion luminescent nano material in 4mL of chloroform solution, dropwise adding the solution into the polyacrylic acid and the absolute ethyl alcohol solution, reacting for 24 hours under magnetic stirring, and centrifugally collecting the material to obtain the modified up-conversion luminescent nano material.

3. The method for preparing the test strip of upconverter aptamer for rapid detection of ochratoxin a as claimed in claim 1 or 2, wherein: the preparation of the up-conversion aptamer probe comprises the steps of dispersing 20mg of the modified up-conversion luminescent nano material in 4mL of 10mM morpholine ethanesulfonic acid buffer solution, carrying out ultrasonic dispersion, adding 0.2mL of 5 mg/mLN-hydroxy thiosuccinimide and 0.1mL of 5mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution, oscillating for 2 hours in a shaker at 37 ℃ for carboxyl activation, dispersing in 2mL of aqueous solution after centrifugal washing, adding an amination aptamer, incubating for 24 hours at 37 ℃, and dispersing in 2mL of water after centrifugal washing to obtain the up-conversion aptamer probe.

4. The method for preparing the test strip of upconverter aptamer for rapid detection of ochratoxin a as claimed in claim 1 or 2, wherein: the aptamer sequence is Apt_{OTA 1}：5’-NH₂-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGATCGGGTGTGGGTGGCGTAAAG GGAGCATCGGACA-3'; the aptamer recognition sequence and the complementary sequence DNA of the non-recognition sequence_OTAThe sequence of the DNA is 5' -bio-TGTCCGATGCTCCCTTTACGCCACCCACACCCG ATC-3_polyAThe sequence is 5 '-bio-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3'.