CN113980682B - Preparation of near-infrared luminescent Yb and Tm co-doped core-shell structure nanocrystalline and application thereof in immunochromatography detection - Google Patents

Preparation of near-infrared luminescent Yb and Tm co-doped core-shell structure nanocrystalline and application thereof in immunochromatography detection Download PDF

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CN113980682B
CN113980682B CN202111354257.3A CN202111354257A CN113980682B CN 113980682 B CN113980682 B CN 113980682B CN 202111354257 A CN202111354257 A CN 202111354257A CN 113980682 B CN113980682 B CN 113980682B
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冀天星
魏嵬
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Second Affiliated Hospital of Guangzhou Medical University
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Abstract

The invention belongs to the technical field of immunology detection, and particularly relates to preparation of near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystals and application thereof in immunochromatography detection; meanwhile, the doping proportion of the luminescent ions Tm of the intermediate shell layer is determined to be 2%. The balance of cross relaxation quenching phenomenon and energy effective transfer between Yb and Tm is realized in the proportion, and finally, the near infrared luminous intensity of the core-shell structure nanocrystalline is greatly improved (850 times of the luminous intensity of standard Yb and Tm bare core nanocrystalline is improved). Then, the high-brightness near-infrared luminescent nanocrystalline is used as an immunofluorescence probe to establish an immunochromatography detection method, so that the immunochromatography method for detecting the disease index with high sensitivity and without background interference is realized.

Description

Preparation of near-infrared luminescent Yb and Tm co-doped core-shell structure nanocrystalline and application thereof in immunochromatography detection
Technical Field
The invention belongs to the technical field of immunological detection, and particularly relates to preparation of near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystals and application thereof in immunochromatography detection.
Background
In recent years, with the continuous development of ambulatory and home medicine, researchers have continuously sought new luminescent materials as luminescent probes to achieve high sensitivity and high accuracy in measuring various disease markers in serum, including myocardial infarction markers (CTNI and MYO); bacterial viral infection markers (CRP, SAA and PCT); diabetes markers (glycosylated hemoglobin); preoperative infectious disease markers (HIV, syphilis, hepatitis b and c), and sudden infectious diseases. The detection method based on the gold nanoparticles has fast development, and has been rapidly applied in the fields of pregnancy detection, infection, sudden infectious disease detection and the like. However, the low-sensitivity nano-gold chromatography detection method belongs to semi-quantitative detection and is not suitable for the application of high-sensitivity detection of disease indexes. The use of fluorescent microspheres and quantum dots improves the sensitivity of the detection method and realizes quantitative detection, but the fluorescent background signal is high in the test process, thereby limiting the application of the fluorescent microspheres and quantum dots in the detection of low-concentration objects to be detected. The rare earth up-conversion nanocrystalline is used as a new generation luminescent material, has large anti-stoke luminescence displacement and narrow emission spectrum, and can realize multi-index multi-detection; the bleaching performance is not realized, the luminous stability is good, and repeated detection can be carried out for many times; most of the excitation light is positioned at 980 or 800nm, and can not excite biological organic molecules such as antigens or antibodies, and the like, so that detection with no background interference and high signal-to-noise ratio can be realized.
At present, typical rare earth up-conversion nanocrystalline is core-shell Yb and Tm co-doped nanocrystalline. However, the Yb and Tm co-doped nanocrystalline with the traditional core-shell structure is a Yb and Tm bare core nanocrystalline, and the luminous intensity of the Yb and Tm co-doped nanocrystalline is obviously lower than that of luminous materials such as quantum dots, fluorescent microspheres and the like. Therefore, improving the luminous intensity of the Yb and Tm co-doped nanocrystals with core-shell structures is a hot spot and an important point of research. For example, the Chinese patent application ZL201810629698.1 provides an improved Yb and Tm co-doped nanocrystalline with a core-shell structure, but the near infrared luminescence of the nanocrystalline is only 4 times higher than that of standard Yb and Tm bare-core nanocrystalline. And the sensitized ion Yb high doping of the core-shell structure nanocrystalline adopted by the patent is extremely easy to cause cross relaxation fluorescence quenching phenomenon, so that the capability of absorbing 980nm photons is not obviously improved. Therefore, the core-shell structure nanocrystal needs to be further improved to further improve the absorption capacity of the nanocrystal to 980nm light, so as to realize high-sensitivity real-time detection of zero background signal interference of an immunochromatography detection method.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a preparation method of near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystalline, which synthesizes NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The core-shell structure nanocrystalline greatly improves the absorption capacity of 980nm light by the nanocrystalline, and is established by taking the core-shell structure nanocrystalline as an immunofluorescence probeThe immunochromatography detection method realizes the immunochromatography method for detecting the disease index with high sensitivity and without background interference.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a preparation method of near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystals, which comprises the following steps:
s1, preparing nuclear NaYF 4 ：
S11, firstly mixing oleic acid with octadecene, then adding yttrium acetate tetrahydrate, heating to 150-180 ℃ and reacting for 120min in vigorous stirring, and cooling to obtain a mixed solution;
s12, adding a methanol solution of sodium hydroxide and ammonium fluoride into the mixed solution in the step S11, heating to 120 ℃ and keeping for 30min, and then heating to 300-320 ℃ under the condition of introducing nitrogen to react for 60-90min;
s13, cooling, washing and centrifuging the reacted mixed solution to obtain beta-NaYF 4 beta-NaYF 4 Dispersing in n-hexane to obtain beta-NaYF 4 A dispersion;
s2, core-shell NaYF 4 @NaF 4 Synthesis of Yb98%, tm 2%:
s21, the mass percentage of ytterbium oxide and thulium oxide is 98 percent: 2% of ytterbium oxide and thulium oxide are dissolved in trifluoroacetic acid water solution, heated to be completely dissolved at 90-100 ℃, then continuously heated to be completely dried and cooled to room temperature;
s22, adding sodium trifluoroacetate, oleic acid and octadecene into the step S21, and preparing the beta-NaYF in the step S13 4 Heating the dispersion to 120-150 ℃ for 30-60min, then heating to 300 ℃ for reacting for 60min under the condition of introducing nitrogen, and growing a shell layer;
s23, cooling, washing and centrifuging the solution obtained in the step S22 to obtain the core-shell NaYF 4 @NaF 4 Yb98%, tm2% and core-shell NaYF 4 @NaF 4 Yb98% and Tm2% are dispersed in n-hexane to obtain core-shell NaYF 4 @NaF 4 Yb98%, tm2% dispersion;
S3、NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 preparing core-shell structure nanocrystalline:
s31, adding yttrium oxide into trifluoroacetic acid aqueous solution, heating to be completely dissolved at 90-100 ℃, then continuously heating to be completely dried, and cooling to room temperature;
s32, adding sodium trifluoroacetate, oleic acid and octadecene into the step S31 and preparing the core-shell NaYF from the step S23 4 @NaF 4 Heating Yb98%, tm2% dispersion liquid, sodium trifluoroacetate, oleic acid and octadecene to 120-150 ℃ for 30-60min, then heating to 300-320 ℃ for 60-90min for growing a shell layer;
s33, cooling, washing and centrifuging the solution in the step S32 to obtain near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystalline, namely NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 Core-shell structure nanocrystals.
Preferably, in step S31, the concentration of yttrium oxide in the aqueous trifluoroacetic acid solution is 0.5-2mmol/6mL. Specifically, the concentration of yttrium oxide in the trifluoroacetic acid aqueous solution is 1mmol/6mL.
Preferably, in step S32, the sodium trifluoroacetate, oleic acid, octadecene and core-shell NaYF 4 @NaF 4 Yb98%, tm2% dispersion with a dosage ratio of 1mmol:10-20mL:10-20mL:1-10mL. Specifically, the sodium trifluoroacetate, oleic acid, octadecene and core-shell NaYF 4 @NaF 4 Yb98%, tm2% dispersion with a dosage ratio of 1mmol:15mL:15mL:5mL.
Preferably, in step S21, the total amount of ytterbium oxide and thulium oxide used is 0.5-2mmol/6mL in trifluoroacetic acid aqueous solution. Specifically, the total dosage of ytterbium oxide and thulium oxide is 1mmol/6mL in trifluoroacetic acid aqueous solution.
Preferably, in step S22, the sodium trifluoroacetate, oleic acid, octadecene and beta-NaYF 4 The dosage ratio of the dispersion was 1mmol:10-20mL:10-20mL:1-10m L. In particular, sodium trifluoroacetate, oleic acid, octadecene and beta-NaYF 4 The dosage ratio of the dispersion was 1mmol:15mL:15mL:5mL.
Preferably, in step S11, the dosage ratio of oleic acid, octadecene and yttrium acetate tetrahydrate is 5-10mL:10-20mL:0.5-5mmol. Specifically, the dosage ratio of oleic acid, octadecene and yttrium acetate tetrahydrate is 7.5mL:15mL:1mmol.
Preferably, in step S12, the volume ratio of the methanol solution of sodium hydroxide and ammonium fluoride to octadecene is 1-3:3. Specifically, the volume ratio of the methanol solution of sodium hydroxide and ammonium fluoride to the octadecene is 2:3.
Preferably, the aqueous solution of trifluoroacetic acid according to the present invention has a concentration of 50% by volume of trifluoroacetic acid.
The invention also provides the near infrared luminescent Yb and Tm codoped core-shell structure nanocrystalline prepared by the preparation method of the near infrared luminescent Yb and Tm codoped core-shell structure nanocrystalline.
The invention also provides application of the near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystal in immunochromatography detection.
The invention also provides a serum marker luminescence detection side flow test strip based on near infrared excitation and emission, which is a side flow test strip taking the near infrared luminescence Yb and Tm co-doped core-shell structure nanocrystal as an immune marker, wherein the side flow test strip consists of a near infrared luminescence Yb and Tm co-doped core-shell structure nanocrystal, a polyvinyl chloride supporting backboard, a detection pad, a combination pad, a sample pad and a water absorption pad, and the combination pad is loaded with a combination antibody or a near infrared luminescence Yb and Tm co-doped core-shell structure nanocrystal probe modified by target protein and a near infrared luminescence Yb and Tm co-doped core-shell structure nanocrystal probe modified by goat anti-chicken IgY antibody.
Preferably, the preparation method of the near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystalline probe modified by the conjugated antibody or the target protein and the near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystalline probe modified by the goat anti-chicken IgY antibody comprises the following steps:
(1) NaYF is prepared 4 @NaYF 4 :Yb,Tm@NaYF 4 The core-shell structure nanocrystalline is dispersed in normal hexane to obtain NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 Nanocrystalline dispersion liquid with core-shell structure, naYF 4 @NaYF 4 :Yb,Tm@NaYF 4 Mixing and oscillating the core-shell structure nanocrystalline dispersion liquid and an HCL solution (0.1M) and ethanol to obtain a mixed liquid; the solution was then sonicated at room temperature for 30min to remove oleic acid ligand. After the reaction is finished, centrifugally collecting NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 Core-shell structure nanocrystals (12000 r,20 min), and re-dispersed in 10mL deionized water after washing 2 times with deionized water;
(2) NaYF to be dispersed in water 4 @NaYF 4 :Yb,Tm@NaYF 4 Dropwise adding the core-shell structure nanocrystalline into deionized water containing PAA, and stirring at room temperature for 60min to obtain PAA encapsulated NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 Core-shell structure nanocrystals. Centrifugation to collect PAA-encapsulated NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 The core-shell structure nanocrystalline is re-dispersed in 10mL deionized water after being washed 2 times by deionized water;
(3) NaYF encapsulating PAA 4 @NaYF 4 :Yb,Tm@NaYF 4 The core-shell structure nanocrystalline is washed three times by MES buffer solution (PH 5.0-8.0) and then is redispersed in the MES buffer solution. Then MES buffer solution (pH 5.0-8.0) containing Sulfo-NHS and EDC was added and slowly shaken for 30min to allow NaYF to pass 4 @NaYF 4 :Yb,Tm@NaYF 4 The core-shell structure nanocrystalline is activated by Sulfo-NHS-and finally is redispersed in MES buffer solution after being washed three times by MES buffer solution.
(4) Adding protein or antibody such as RBD or chicken IgY to the NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 Slowly shaking for 60 minutes in a core-shell structure nanocrystalline solution to obtain protein or antibody coupled NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 Core-shell structure nanocrystals. Then continuously adding a labeling and blocking solution (0.04M ethanolamine, 1% (w/v) Bovine Serum Albumin (BSA) aqueous solution), slowly shaking for 30 minutes to block other activation sites, and centrifuging to obtain a near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystalline probe combined with an antibody or modified by target protein and modified by a goat anti-chicken IgY antibodyNear infrared luminescent Yb and Tm co-doped core-shell structure nanocrystalline probes.
Compared with the prior art, the invention has the beneficial effects that:
the invention adopts the synthesis scheme of the core-shell structure nanocrystalline, and the high doping of sensitized ion Yb to 98% can be realized by using the scheme, so that the absorption capacity of the nanocrystalline to 980nm light is improved; meanwhile, the doping proportion of the luminescent ions Tm of the intermediate shell layer is determined to be 2%. The balance of cross relaxation quenching phenomenon and energy effective transfer between Yb and Tm is realized in the proportion, and finally, the near infrared luminous intensity of the core-shell structure nanocrystalline is greatly improved (850 times of the luminous intensity of standard Yb and Tm bare core nanocrystalline is improved). Then, the high-brightness near-infrared luminescent nanocrystalline is used as an immunofluorescence probe to establish an immunochromatography detection method, so that the immunochromatography method for detecting the disease index with high sensitivity and without background interference is realized. Specifically, the invention has the following advantages:
(1) The invention synthesizes NaYF by doping Yb and Tm rare earth elements 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 Core-shell structured nanocrystals (hexagonal phase). The Yb absorbs 980nm photons, and the energy transfer mode of Yb-Tm is utilized to make Tm emit 800nm fluorescence. The invention uses the inner core and the outermost inert shell NaYF 4 Sandwich interlayer NaF 4 The core-shell structure formed by Yb98% and Tm2% can limit excitation energy in the intermediate layer and inhibit cross relaxation quenching phenomenon caused by high-concentration Yb doping, and finally the NaYF with high-brightness near-infrared luminescence is obtained 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 Rare earth up-conversion luminescence nanocrystalline;
(2) The invention provides a near infrared excitation and emission up-conversion luminescent material NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The surface contains a large amount of oleic acid ligand, the oleic acid modified nanocrystal is converted into water solubility from oil solubility by utilizing a plurality of carboxyl groups contained in PAA, then the exposed carboxyl groups on the surface of the nanocrystal are subjected to activation treatment by EDC/NHS, so that the carboxyl groups are covalently connected with amino groups of protein or antibody, and the protein or antibody is coupled to the nanocrystal to obtain the protein or antibodyNaYF modified by human antibody 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The nanocrystalline probe is used as an immune mark to prepare a serum marker luminescent detection side flow test strip, and finally a serum marker (myoglobin, troponin, procalcitonin, glycosylated hemoglobin, HIV, hepatitis B, hepatitis C, novel coronavirus and the like) immune chromatography detection method based on near infrared light excitation and near infrared detection is developed.
(3) When the lateral flow test strip is used for detecting the actual body fluid marker, a certain amount of body fluid sample is dripped on a sample pad, after hatching for a certain time, a portable detection instrument (980 nm excitation, 800nm detection; chinese patent 202021494870.6) is used for detecting the luminescence of the test strip. Due to NaYF-based 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The excitation and emission spectrums of the luminescent probe of the nanocrystalline are both in the near infrared region, so that the interference of various small molecules and large molecular substances in cellulose films and body fluids on detection fluorescent signals is eliminated, and the high-sensitivity real-time detection of zero background signal interference of an immunochromatography detection method is realized.
Drawings
FIG. 1 is an XRD diffraction pattern of a core-shell structure NaYF4@NaF4:98% Yb,2% Tm@NaYF4;
FIG. 2 is a transmission electron microscope image of a core-shell structure NaYF4@NaF4:98%Yb,2%Tm@NaYF4 (a bar graph indicates the size of the core-shell structure nanocrystal);
FIG. 3 is a core-shell NaYF structure 4 @NaF 4 :98％Yb,2％Tm@NaYF 4 Compared with the conventional NaYF 4 A fluorescence spectrum comparison chart of 20% Yb and 1% Tm nanocrystalline;
FIG. 4 is a graph showing the comparison of fluorescence spectra of Yb and Tm co-doped nanocrystals of core-shell structure disclosed in patent ZL 201810629698.1;
FIG. 5 is a graph of NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 The core-shell structure nanocrystalline is a schematic diagram of a lateral flow test strip marked by immunofluorescence ((1) is a sample pad, (2) is a bonding pad, (3) is a T line, (4) is a C line, and (5) is a water absorption pad);
FIG. 6 is a graph of NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 The core-shell structure nanocrystalline is a physical image of a lateral flow test strip marked by immunofluorescence;
FIG. 7 is a schematic representation of NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 The core-shell structure nanocrystalline is a graph of relation between SARS-CoV-2RBD-IgG concentration in serum sample measured by immunofluorescence marked side flow test strip and 800nm fluorescence intensity of detection line and quality control line;
FIG. 8 shows the reaction of NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 The core-shell structure nanocrystalline is a regression analysis result graph of the detection result of the side flow test strip marked by immunofluorescence and the detection result of the Roche chemiluminescent detector (ELISA method is used as a contrast);
FIG. 9 shows the reaction of NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 The core-shell structure nanocrystalline is an emission spectrum chart of a lateral flow test strip detection line marked by immunofluorescence (ELISA method is used as a control).
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
Example 1 near infrared luminescent Yb and Tm Co-doped core-shell Structure nanocrystalline (NaYF) 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 Nanocrystalline with core-shell structure)
1. Preparation of Nuclear NaYF 4
1) 7.5mL of Oleic Acid (OA) and 15mL of Octadecene (ODE) were added to a three-necked flask followed by 1mmol of YAc 3 ，4H 2 O (yttrium acetate tetrahydrate) and then heating to 160 ℃ for 120min to completely dissolve so as to remove the OWater and oxygen, and finally cooling to room temperature to obtain a mixed solution;
2) 10mL of the mixture contains NH 4 Adding a methanol solution of F (1 mmoL) and NaOH (1 mmoL) into the mixed solution of the three-neck flask, and heating to 120 ℃ for 30min to remove methanol; then introducing enough nitrogen and heating the mixture to 300 ℃ for reaction for 60min;
3) After the reaction is finished, naturally cooling the obtained liquid to below 100 ℃, transferring the liquid into a centrifuge tube, adding 5mL of absolute ethyl alcohol, ultrasonically vibrating the centrifuge tube, centrifuging for 5min at a speed of 12000r/min, and repeating the operations of ethanol vibration washing and centrifugal separation for 2-3 times to obtain nuclear NaYF 4 。
Finally, washing and centrifuging are repeated 3 times by using absolute ethyl alcohol, and then the nuclear beta-NaYF is obtained 4 Dispersing in 5mL cyclohexane to obtain nuclear NaYF 4 And (3) dispersing the solution for later use.
2. Core-shell NaYF 4 @NaF 4 Synthesis of Yb98%, tm2%
1) Adding 0.98mmol ytterbium oxide and 0.02mmol thulium oxide to a mixture containing 3mLH 2 In a three-necked flask containing O and 3mL of trifluoroacetic acid, the flask was heated to 98℃until it was completely transparent, and the entire flask was dissolved. Then, the mixture was directly heated to remove all the water, and the white powder at the bottom of the three-necked flask was completely dried and cooled to room temperature.
2) Into a three-necked flask, 1mmol of sodium trifluoroacetate, 15mLOA,15mLODE and 5mL of the β -NaYF of step 1 were continuously charged 4 The dispersion was warmed to 120 ℃ to remove water and cyclohexane. Then the temperature is raised to 300 ℃ for reaction for 60min under the condition of sufficient nitrogen for growing the shell.
3) After the solution obtained in the step 2) is naturally cooled to room temperature, adding 10mL of absolute ethyl alcohol for vibration washing, centrifuging for 5min at 12000r/min, and repeating the operation of the vibration washing of the ethyl alcohol and the centrifugal separation for 2-3 times to obtain the core-shell NaYF 4 @NaYbF 4 .Tm。
The prepared core-shell NaYF4@NaYbF is prepared 4 Tm is dispersed in 3mL cyclohexane to prepare core-shell NaYF4@NaYbF 4 Tm dispersion.
3、NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 Preparation of core-shell structure nanocrystalline
1) 1mmol of yttria was added to a mixture containing 3mLH 2 Heating to 98 ℃ in a three-neck flask containing O and 3mL of trifluoroacetic acid until the mixture is completely transparent, so that the mixture is completely dissolved; then, continuing heating until all water is removed, until the white powder (namely the trifluoroacetic acid rare earth salt) can be obtained at the bottom of the three-neck flask after the white powder is completely dried, and cooling to room temperature;
2) Into a three-necked flask, 1mmol of sodium trifluoroacetate, 15mLOA,15mLODE and 5mL of NaYF prepared in step 2 were continuously added 4 @NaF 4 Yb98 percent, tm2 percent of dispersion liquid, heating to 120 ℃ and keeping for 45 minutes to remove water and cyclohexane, and heating to 300 ℃ to react for 60 minutes under the condition of introducing nitrogen for growing a shell layer;
3) After the solution obtained in the step 2) is naturally cooled to room temperature, adding 10mL of absolute ethyl alcohol for vibration washing, centrifuging for 5min at 12000r/min, and repeating the operation of the vibration washing of the ethyl alcohol and the centrifugal separation for 2-3 times to obtain NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 Core-shell structure nanocrystals.
The NaYF obtained was then subjected to 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The nano-crystal with the core-shell structure is dispersed in cyclohexane to prepare NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 And (3) core-shell structured nanocrystalline dispersion liquid for standby.
For NaYF prepared in this example 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 XRD diffraction analysis is carried out on the nanocrystalline with the core-shell structure. As can be seen from the XRD diffraction pattern shown in FIG. 1, naYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The nanocrystalline is a hexagonal phase structure;
for NaYF prepared in this example 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 And (5) carrying out transmission electron microscope observation on the nanocrystalline with the core-shell structure. As can be seen from FIG. 2, the core-shell NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The nanocrystalline is monodisperse, has uniform particle size and round particles, and has a diameter of 30nm;
for NaYF prepared in this example 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 Nanocrystalline with core-shell structure and beta-NaYF with core-shell structure 4 Analysis of emission spectra at 980nm excitation of 20% Yb,1% Tm (standard Yb and Tm bare core nanocrystals) (test using portable detection instrument as disclosed in Chinese patent No. 202021494870.6). As shown in FIG. 3, by NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The near infrared luminescence of the nanocrystalline at 800nm is enhanced by 850 times. And the near infrared luminescence of the Yb and Tm co-doped nanocrystalline with the core-shell structure disclosed in the patent ZL201810629698.1 is only improved by 4 times compared with that of the standard Yb and Tm bare-core nanocrystalline (shown in fig. 4).
Wherein the above mentioned core-shell structure beta-NaYF 4 The preparation method of 20% Yb and 1% Tm is as follows:
1) 7.5mL of Oleic Acid (OA) and 15mL of Octadecene (ODE) were added to a three-necked flask followed by 0.79mmol of YAc 3 ，4H 2 O (yttrium acetate tetrahydrate), 0.2mmolYbAc 3 ,4H 2 O (ytterbium acetate tetrahydrate), 0.01mmol TmAc 3 ,4H 2 O (thulium acetate tetrahydrate), heating to 160 ℃ to react for 120min until the O is completely dissolved so as to remove moisture and oxygen in the O, and finally cooling to room temperature to obtain a mixed solution;
2) 10mLNH was used 4 Adding a methanol solution of F (1 mmoL) and NaOH (1 mmoL) into the mixed solution of the three-neck flask, and heating to 120 ℃ for 30min to remove methanol; then introducing enough nitrogen and heating the mixture to 300 ℃ for reaction for 60min;
3) After the reaction is finished, naturally cooling the obtained liquid to below 100 ℃, transferring the liquid into a centrifuge tube, adding 5mL of absolute ethyl alcohol, ultrasonically vibrating the centrifuge tube, centrifuging the centrifuge tube for 5min at a speed of 12000r/min, and repeating the ethanol vibration washing and centrifugal separation operation for 2-3 times to obtain the beta-NaYF with a core-shell structure 4 20% Yb,1% Tm. Finally, washing and centrifuging with absolute ethyl alcohol repeatedly for 3 times, and then carrying out beta-NaYF on the core-shell structure 4 20% Yb and 1% Tm are dispersed in 5mL cyclohexane to obtain the core-shell structure beta-NaYF 4 20% Yb,1% Tm dispersion.
Example 2 preparation of a detection lateral flow strip for luminescent detection of serum markers based on near-infrared excitation and emission
The lateral flow test strip of the embodiment is a lateral flow test strip which uses NaYF4@NaYF4 of the embodiment 1, wherein the NaYF4@NaYF4 core-shell structure nanocrystalline is an immunolabeling lateral flow test strip, and the lateral flow test strip consists of NaYF4@NaYF4:Yb, tm@NaYF4 core-shell structure nanocrystalline, a polyvinyl chloride supporting backboard, a detection pad, a binding pad, a sample pad and a water absorption pad (purchased by whatman company);
the detection pad consists of a nitrocellulose membrane, and a detection line and a control line which are drawn on the nitrocellulose membrane in parallel; the detection line is a line drawn by serum index antibody with the concentration of 1-2 mg/mL, the line width is 1-1.2 mm, and the dropwise adding volume is 1-1.2L/cm; the control line is a line drawn by using 1-2 mg/mL sheep anti-chicken IgY antibody solution, the line width is 1-1.2 mm, and the dropping volume is 1-1.2L/cm;
the binding pad is loaded with a binding antibody or target protein modified NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 Core-shell structure nanocrystalline probe and sheep anti-chicken IgY antibody modified NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 Core-shell structured nanocrystalline probes.
The detection pad is stuck to the middle part of the polyvinyl chloride support backboard, and is lapped with the bonding pad on one side of a detection line (T line) of the detection pad; the other end of the bonding pad is overlapped with the sample pad; the water absorption pad is lapped with the detection pad at one side of a control line (C line) of the detection pad; the conjugate pad, sample pad and absorbent pad were also attached to a polyvinyl chloride support backing (the construction of which is shown in fig. 5 and 6).
The preparation method of the lateral flow test strip comprises the following steps:
1. preparation of binding antibodies or target protein modified NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 Core-shell structure nanocrystalline probe and sheep anti-chicken IgY antibody modified NaYF 4 @NaYF 4 :Yb,Tm@NaYF 4 Core-shell structure nanocrystalline probe (namely, rare earth up-conversion luminescence probe preparation)
1) 5mL of HCL solution (0.1M) and 5mL of ethanol were added to NaYF obtained in example 1 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 Core-shell structured nanocrystals (10 mL); the solution is then subjected toRemoving oleic acid ligand by ultrasonic treatment at room temperature for 30 min; centrifuging (12000 r,20 min) after the reaction to obtain NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 A nanocrystalline; washing with deionized water for 2 times, and concentrating NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The nanocrystals were dispersed in 10mL deionized water; then NaYF dispersed in water 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The nanocrystals were added drop-wise to 10mL deionized water in which 50mg PAA (polyacrylic acid) was dissolved; stirring at room temperature for 60min to obtain PAA-encapsulated NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The method comprises the steps of carrying out a first treatment on the surface of the Centrifugation to collect PAA-encapsulated NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 (PAA coated nanocrystalline for short), and after deionized water is washed for 2 times, the PAA coated nanocrystalline water dispersion is dispersed in 10mL of deionized water again to prepare PAA coated nanocrystalline water dispersion;
2) Adding 200 mu LPAA coated nanocrystalline aqueous dispersion into an EP tube, then adding 1mLMES buffer (pH 6.0) for resuspension, centrifuging for 15min at 14,000Xg, and discarding the supernatant; re-suspending the PAA coated nanocrystalline in 1mLMES buffer after repeating for 3 times; then 10mL of MES buffer containing 1M Sulfo-NHS and 1M EDC was added; and slowly shaking for 30 minutes; after washing the sulfoNHS activated nanocrystals 3 times with MES buffer, they were redispersed in MES buffer (1 mL). Subsequently, 120 mg of RBD protein (novel coronavirus antigen) or chicken IgY (purchased from Hangzhou Kogyo Biotechnology Co., ltd.) was added and gently shaken for 60 minutes;
then, 1mL of a labeled blocking solution [ aqueous solution containing 0.04M ethanolamine, 1% (w/v) Bovine Serum Albumin (BSA) ] was added, and the mixture was slowly shaken for 30 minutes to block the other activation sites. After centrifugation (14,000Xg centrifugation for 15 min), the RBD or chicken IgY coupled nanocrystalline probe, namely RBD or chicken IgY coupled NaYF, is obtained 4 @NaYF 4 :Yb,Tm@NaYF 4 Core-shell structured nanocrystalline probes.
Finally, protein or antibody conjugated NaYF was re-conjugated after three washes with labeled final wash [ 10mM Tris-base buffer, containing 1% (w/v) bovine serum albumin, 1% (v/v) TX-100,0.1% (v/v) Tween-20,0.3% (v/v) Proclin-300, pH=8.0 ] 4 @NaYF 4 :Yb,Tm@NaYF 4 The core-shell structure nanocrystalline is dispersed in 200 mu L of marked final washing liquid to form RBD or chicken IgY coupled nanocrystalline probe dispersion liquid (the concentration is 1-2 mg/mL) for standby.
2. Assembled lateral flow test strip
1) Soaking the glass fiber membrane in PB buffer solution containing 1% (v/v) Tween-20 for 2-4 h; then placing the mixture into a blast drying oven at 45 ℃ for drying for 24 hours; cutting the dried glass fiber film into strips with the width of 17mm and the length of 300mm by using an automatic cutter to obtain a standby sample pad;
2) Soaking the polyester fiber membrane in PB buffer solution containing 1% (v/v) Tween-20 for 2-4 h; then placing the mixture into a blast drying oven at 45 ℃ for drying for 24 hours; cutting the dried polyester fiber film into long strips with the width of 8mm and the length of 300mm by using an automatic cutting instrument to obtain a standby bonding pad;
3) A nitrocellulose membrane was stuck to the middle of a polyvinyl chloride support back plate (78 mm. Times.300 mm. Times.0.33 mm); sticking a standby bonding pad on a polyvinyl chloride support backboard at one side of a detection line of the nitrocellulose membrane, and overlapping the nitrocellulose membrane and the standby bonding pad by 2mm; the standby sample pad is stuck on the polyvinyl chloride supporting backboard, and the standby sample pad is overlapped with the standby bonding pad for 2mm; on one side of a quality control line of the nitrocellulose membrane, a water absorption pad is stuck on a polyvinyl chloride supporting backboard, and the water absorption pad is lapped with the nitrocellulose membrane for 2mm (a schematic diagram and a physical diagram are respectively shown in fig. 5 and 6);
4) Adding 5% (w/v) sucrose, 5% (w/v) trehalose, 0.3% (w/v) bovine serum albumin, 0.5% (v/v) PVP (K-30), 5% (v/v) S9 (S9 surfactant), 0.3% (v/v) Proclin-300, 8% (v/v) RBD coupled nanocrystalline probe dispersion, 3% (v/v) chicken IgY coupled nanocrystalline probe dispersion into 20mM Tris-base buffer as solvent; spraying the film onto a standby bonding pad in the polyvinyl chloride supporting backboard by using a metal spraying film drawing instrument at a spraying speed of 50mm/s and a spraying amount of 4 mu L/cm;
5) A1 mg/mL solution of a murine anti-human IgG antibody (purchased from Hangzhou Kogyo Biotechnology Co., ltd.) and a 1mg/mL solution of a goat anti-chicken IgY antibody (purchased from Hangzhou Kogyo Biotechnology Co., ltd.) were respectively dispensed on the detection line and the quality control line of the nitrocellulose membrane of the polyvinyl chloride support back plate with a gold spraying film dispenser at a film dispensing speed of 50mm/s and a film dispensing amount of 1. Mu.L/cm. Placing the PVC rubber plate after film drawing in a blast drying oven at 45 ℃ for drying for 12-24 hours;
6) Cutting the dried test paper into a test paper strip with the width of 3.98 mm by an automatic cutter, and filling the test paper strip into a plastic card shell (shown in figure 2), thus preparing the serum marker luminescent detection side flow test paper strip based on near infrared excitation and emission, namely the nano-crystalline SARS-CoV-2RBD-IgG determination test paper strip with the Yb and Tm co-doped core-shell structure.
Prepared in this example as NaYF 4 @NaF 4 :Yb98％,Tm2％@NaYF 4 The using method of the lateral flow test strip with the core-shell structure nanocrystalline as the immunofluorescence mark comprises the following steps: 20 microliters and 80 microliters of buffer [ 20mM Tris-base,0.85% (w/v) NaCl,0.5% (w/v) TX-100,0.1% (v/v) Tween-20, 0.1% (w/v) S9, pH 8.0 ] are added to the sample pad, respectively, and the mixture is allowed to stand for 15 minutes, and then a portable detection instrument (see patent 202021494870.6) is used for detecting fluorescent signals of detection lines and control lines in the test strip at 800 nm; then, a standard curve is established according to the measured luminous intensity and the concentration of a standard sample SARS-CoV-2RBD-IgG antibody (purchased from the offshore protein technology Co., ltd.); comparing the fluorescent signal of the detection sample with a standard curve to obtain the concentration of SARS-CoV-2RBD-IgG antibody in the sample;
example 3 detection limit of near-infrared excitation and emission based serum marker luminescent detection lateral flow strip
The detection limit of SARS-CoV-2RBD-IgG antibody is determined by exploring the lateral flow test strip prepared in example 2, and the specific method is as follows:
1) The SARS-CoV-2RBD-IgG antibody standard (purchased from near shore protein technology Co.) is added into serum sample without SARS-CoV-2RBD-IgG antibody before epidemic situation to prepare a series of serum samples with concentration of SARS-CoV-2RBD-IgG antibody of 0ug/mL,0.05ug/mL,0.1ug/mL,1ug/mL,5ug/mL,10ug/mL,20 ug/mL;
2) 20 microliters of serum samples at each concentration were added to the test paper card loading well (lateral flow strip of example 2), followed by 80 microliters of sample Tris buffer [ 20mM Tris-base,0.85% (w/v) NaCl,0.5% (w/v) TX-100,0.1% (v/v) Tween-20, 0.1% (w/v) S9, pH 8.0 ]. After standing for 15min, carrying out 800nm emission light test on the detection line and the control line of the lateral flow test strip by using a portable detection instrument (Chinese patent 202021494870.6), and collecting the emission light signal intensity of the detection line at 800nm, wherein a bar graph of SARS-CoV-2RBD-IgG antibody concentration and test result of each sample is shown in FIG. 7. As can be seen from FIG. 7, the detection limit of the lateral flow test strip is 0.016. Mu.g/mL.
Example 4 comparison of Positive detection Rate of near-infrared excitation and emission-based serum marker luminescent detection lateral flow strip (NIR-LFA method for short) and ELISA method in determination of clinical blood sample of patient recovering from COVID-19
1) Collecting 16 parts of normal human serum before epidemic situation and 35 parts of serum of a patient recovering from the COVID-19;
2) 50ng of RBD coated ELISA plates were added to each well; blocking the ELISA plates with phosphate buffered saline buffer (PBST) containing 5% milk; the elisa plate was washed 2 times with phosphate buffered saline buffer containing 0.1% tween-20. The diluted plasma (1:200) was then added to the test wells of the ELISA plate at 100. Mu.L per well and incubated at 37℃for 1 hour. The ELISA plate was washed 4 times with PBST and then 100. Mu.L of diluted anti-human IgG antibody (1:8000) per well was added to the ELISA plate and incubated at 37℃for 1 hour. The ELISA plate was washed 4 times with PBST, 50. Mu.L of TMB (3, 3', 5' -tetramethylbenzidine) was added, incubated at room temperature for 5min, and 50. Mu.L of 1M H was added 2 SO 4 The solution stopped the color reaction. Finally, absorbance at 450nm was measured using a Tecan sun micro-plate absorbance meter, switzerland.
3) 20 microliters of serum sample was added to the well of the dipstick (lateral flow dipstick of example 2) and then 80 microliters of sample Tris buffer [ 20mM Tris-base,0.85% (w/v) NaCl,0.5% (w/v) TX-100,0.1% (v/v) Tween-20, 0.1% (w/v) S9, pH 8.0 ] was added; after standing for 15min, carrying out 800nm emission light test on the detection line and the control line of the lateral flow test strip by using a portable detection instrument (Chinese patent 202021494870.6), and collecting the emission light signal intensity of the detection line at 800 nm.
A bar graph of SARS-CoV-2RBD-IgG antibody concentration versus test results for each sample is shown in FIG. 8. As shown in FIG. 8, the detection rate of the nano-crystalline SARS-CoV-2RBD-IgG with the Yb and Tm codoped core-shell structure in clinical blood samples of patients with COVID-19 is obviously higher than that of ELISA method.
Example 5 comparison of the Positive detection Rate of SARS-CoV-2RBD-IgG in blood samples after the measurement of the normal human being vaccinated with SARS-CoV-2 inactivated vaccine by the near-infrared excitation and emission-based serum marker luminescent detection lateral flow strip (abbreviated as NIR-LFA method) and ELISA method
1) Collecting blood samples of 19 normal human serum which is not vaccinated and has no history of COVID-19 disease and 60 normal human which are vaccinated with SARS-CoV-2 inactivated vaccine;
2) 20 microliters of serum sample was added to the loading well of the test paper card (lateral flow strip of example 2), followed by 80 microliters of sample Tris buffer [ 20mM Tris-base,0.85% (w/v) NaCl,0.5% (w/v) TX-100,0.1% (v/v) Tween-20, 0.1% (w/v) S9, pH 8.0 ]. After standing for 15min, carrying out 800nm emission light test on the detection line and the control line of the lateral flow test strip by using a portable detection instrument (Chinese patent 202021494870.6), and collecting the emission light signal intensity of the detection line at 800 nm.
3) 50ng of RBD coated ELISA plates were added to each well; sealing the ELISA plate with phosphate buffer saline containing 5% milk; the elisa plate was washed 2 times with phosphate buffered saline buffer (PBST) containing 0.1% tween-20. Then 100. Mu.L of diluted plasma (1:200) per well was added to the test wells of the ELISA plate and incubated at 37℃for 1 hour. The ELISA plate was washed 4 times with PBST and then added to the ELISA plate in an amount of 100. Mu.L of diluted anti-human IgG antibody (1:8000) per well, and incubated at 37℃for 1 hour. The ELISA plate was washed 4 times with PBST, 50. Mu.LTMB was added, incubated at room temperature for 5min, and 50. Mu.L 1M H was added 2 SO 4 The solution stopped the color reaction. Finally, the absorbance at 450nm was measured by a Tecan Sun microplate absorbance meter, switzerland.
A bar graph of the SARS-CoV-2RBD-IgG antibody concentration and the test result of each sample is shown in FIG. 9, and from FIG. 9, the detection rate of the SARS-CoV-2RBD-IgG in the normal human blood sample after the inactivated vaccine is obviously higher than that of the ELISA method by the Yb and Tm co-doped core-shell structure nanocrystalline SARS-CoV-2RBD-IgG test strip.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.

Claims (8)

1. The application of the near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystalline in immunochromatography detection is characterized in that the preparation method of the near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystalline comprises the following steps:
s1, preparing nuclear NaYF 4 ：
S11, firstly mixing oleic acid with octadecene, then adding yttrium acetate tetrahydrate, heating to 150-180 ℃ and reacting for 120min in vigorous stirring, and cooling to obtain a mixed solution;
s12, adding a methanol solution of sodium hydroxide and ammonium fluoride into the mixed solution in the step S11, heating to 120 ℃ and keeping for 30min, and then heating to 300-320 ℃ under the condition of introducing nitrogen to react for 60-90min;
s13, cooling, washing and centrifuging the reacted mixed solution to obtain beta-NaYF 4 beta-NaYF 4 Dispersing in n-hexane to obtain beta-NaYF 4 A dispersion;
s2, core-shell NaYF 4 @NaYbF 4 Synthesis of Tm:
s21, the mass percentage of ytterbium oxide and thulium oxide is 98 percent: 2% of ytterbium oxide and thulium oxide are dissolved in trifluoroacetic acid water solution, heated to be completely dissolved at 90-100 ℃, then continuously heated to be completely dried and cooled to room temperature;
s22, adding sodium trifluoroacetate, oleic acid and octadecene into the step S21, and preparing the beta-NaYF in the step S13 4 Heating the dispersion to 120-150deg.C, maintaining for 30-60min, and introducing nitrogen gasHeating to 300 ℃ for reacting for 60min for growing a shell layer;
s23, cooling, washing and centrifuging the solution obtained in the step S22 to obtain the core-shell NaYF 4 @NaYbF 4 Tm, core-shell NaYF 4 @NaYbF 4 Tm is dispersed in n-hexane to obtain core-shell NaYF 4 @NaYbF 4 Tm dispersion;
S3、NaYF 4 @NaYbF 4 :Tm@NaYF 4 preparing core-shell structure nanocrystalline:
s31, adding yttrium oxide into trifluoroacetic acid aqueous solution, heating to be completely dissolved at 90-100 ℃, then continuously heating to be completely dried, and cooling to room temperature;
s32, adding sodium trifluoroacetate, oleic acid and octadecene into the step S31 and preparing the core-shell NaYF from the step S23 4 @NaYbF 4 Tm dispersion, sodium trifluoroacetate, oleic acid and octadecene are heated to 120-150 ℃ for 30-60min, then heated to 300-320 ℃ for 60-90min for growing a shell layer;
and S33, cooling, washing and centrifuging the solution in the step S32 to obtain the near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystal.
2. The use of near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystals in immunochromatographic assay according to claim 1, wherein in step S31, the concentration of yttria in aqueous trifluoroacetic acid is 0.5-2mmol/6mL.
3. The use of near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystals in immunochromatographic detection according to claim 1, wherein in step S32, sodium trifluoroacetate, oleic acid, octadecene and core-shell NaYF 4 @NaYbF 4 The use ratio of Tm dispersion was 1mmol:10-20mL:10-20mL:1-10mL.
4. The use of near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystals in immunochromatographic detection according to claim 1, wherein in step S21, the total amount of ytterbium oxide and thulium oxide used is 0.5-2mmol/6mL in trifluoroacetic acid aqueous solution.
5. The use of near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystals for immunochromatographic detection according to claim 1, wherein in step S22, the sodium trifluoroacetate, oleic acid, octadecene and beta-NaYF 4 The dosage ratio of the dispersion was 1mmol:10-20mL:10-20mL:1-10m L.
6. The application of near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystals in immunochromatography detection according to claim 1, wherein in step S11, the dosage ratio of oleic acid, octadecene and yttrium acetate tetrahydrate is 5-10mL:10-20mL:0.5-5mmol.
7. The use of near infrared luminescent Yb and Tm co-doped core-shell structure nanocrystals in immunochromatographic detection according to claim 1, wherein in step S12, the volume ratio of the methanol solution of sodium hydroxide and ammonium fluoride to octadecene is 1-3:3.
8. The serum marker luminescence detection side flow test strip based on near infrared excitation and emission is characterized in that the side flow test strip is a side flow test strip taking the near infrared luminescence Yb and Tm co-doped core-shell structure nanocrystal as an immune label, the side flow test strip consists of a near infrared luminescence Yb and Tm co-doped core-shell structure nanocrystal, a polyvinyl chloride supporting backboard, a detection pad, a binding pad, a sample pad and a water absorption pad, wherein the binding pad is loaded with a binding antibody or a target protein modified near infrared luminescence Yb and Tm co-doped core-shell structure nanocrystal probe and a sheep anti-chicken IgY antibody modified near infrared luminescence Yb and Tm co-doped core-shell structure nanocrystal probe.
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