CN115572596A - Up-conversion nanoparticle group for detecting biomarkers through two channels and preparation method and application thereof - Google Patents

Up-conversion nanoparticle group for detecting biomarkers through two channels and preparation method and application thereof Download PDF

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CN115572596A
CN115572596A CN202210724767.3A CN202210724767A CN115572596A CN 115572596 A CN115572596 A CN 115572596A CN 202210724767 A CN202210724767 A CN 202210724767A CN 115572596 A CN115572596 A CN 115572596A
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孙天瀛
郭铭禹
麻新健
曾兰香
高耀斌
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Sun Yat Sen University
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Abstract

The application belongs to the technical field of fluorescent materials, and particularly relates to an up-conversion nanoparticle group for detecting biomarkers through two channels, and a preparation method and application thereof. The application provides an upconversion nanoparticle set for dual-channel biomarker detection, comprising a first carboxyl-modified upconversion nanoparticle and a second carboxyl-modified upconversion nanoparticle; naYF 4 @NaYbF 4 :Er@NaYF 4 The shell layer is modified with carboxyl; naGdF 4 :Yb/Tm@NaNdF 4 :Yb@NaGdF 4 The shell layer is modified with carboxyl. In the present applicationThe up-conversion nanoparticle group is applied to the detection of the biomarkers, and the biomarkers can be specifically detected while double excitation, double emission and double detection are realized.

Description

Up-conversion nanoparticle group for detecting biomarkers through two channels and preparation method and application thereof
Technical Field
The application belongs to the technical field of fluorescent materials, and particularly relates to an up-conversion nanoparticle group for detecting biomarkers through two channels, and a preparation method and application thereof.
Background
High throughput multiplex immunoassays facilitate the simultaneous detection of multiple analytes in a single sample and reduce assay time and cost. Recently, detection techniques of biomarkers with ultra-high sensitivity have drawn a great deal of attention due to their great role in early disease diagnosis. Clinical diagnostics also increasingly require the measurement of multiple biomarkers for multiple diseases. At present, enzyme-linked immunosorbent assay (ELISA) is still considered as the most mature commercial method for detecting clinical biomarkers and is widely studied. Enzyme-linked immunosorbent assay is divided into direct method, competitive method, double-antibody sandwich method, etc. the application is improved based on double-antibody sandwich enzyme-linked immunosorbent assay. The double-anti sandwich enzyme-linked immunosorbent assay is characterized in that an antigen-antibody complex is formed on a solid phase support such as a microporous plate, an enzyme-labeled antibody is added into the antigen-antibody complex, and enzyme is developed through a substrate to achieve the detection purpose, wherein the process is shown in figure 2, and figure 2 is a schematic diagram of the double-anti sandwich enzyme-linked immunosorbent assay in the prior art. In this technique, the concentration of the antigen is calculated from the absorbance (OD value) of the color developed. However, since the reliability of the conventional catalytic reaction-based ELISA is not satisfactory for ultrasensitive multiplex assays because the stability of the enzyme is limited, color interference may occur and the generation of a signal takes a long time, and in addition, the enzyme is easily inactivated at high temperature, the activity is reduced at low temperature, and the enzyme is also inactivated in the presence of acid, base, and heavy metals, the conventional enzyme-based method is very harsh to assay conditions and reagent storage conditions, and because the ELISA is based on a signal of light absorption, the sensitivity is limited.
Disclosure of Invention
In view of this, the present application provides an upconversion nanoparticle set for dual-channel biomarker detection, and a preparation method and an application thereof, which can realize simultaneous specific biomarker detection of dual excitation, dual emission and dual detection.
The present application provides in a first aspect a set of up-converting nanoparticles for two-channel biomarker detection, comprising:
a carboxyl-modified first upconverting nanoparticle and a carboxyl-modified second upconverting nanoparticle;
the first upconversion nanoparticles are NaYF 4 The nano particles of the matrix are cores, and Er is sequentially coated outside the cores 3+ Doped NaYbF 4 Intermediate shell of nanoparticles of matrix and NaYF 4 Surface shell of nanoparticles of substrate constituting Yb 3+ And Er 3+ Codoped NaYF 4 @NaYbF 4 :Er@NaYF 4 The core-shell nanoparticles of (1);
the second upconversion nanoparticle is doped with Yb 3+ And Tm 3+ With NaGdF 4 The nano particles of the matrix are cores, and Yb is coated outside the cores in sequence 3+ Doped NaNdF 4 Intermediate shell of nanoparticles of a matrix and NaGdF 4 Surface shell of nanoparticles of a matrix constituting Nd 3+ 、Yb 3+ And Tm 3+ Co-doped NaGdF 4 :Yb/Tm@NaNdF 4 :Yb@NaGdF 4 Core-shell nanoparticles of (a);
the NaYF 4 @NaYbF 4 :Er@NaYF 4 The surface of the nano-particles is modified with carboxyl; the NaGdF 4 :Yb/Tm@NaNdF 4 :Yb@NaGdF 4 The surface of the nano-particles is modified with carboxyl.
In another embodiment, the first upconverting nanoparticle fluoresces one color when excited by near infrared 980nm wavelength; the second up-conversion nano-particles emit fluorescence of another color under the excitation of the wavelength of 808nm of near infrared light.
In another embodiment, the Er is present in the core of the first upconverting nanoparticle, in the intermediate shell of the first upconverting nanoparticle, or in a combination thereof 3+ The doping molar concentration of the ions is 2 to 100%, preferably 2 to 20%, more preferably 2%.
In another embodiment, the second upconversion nanoparticle is insideIn the nucleus of said Yb 3+ The doping molar concentration of ions is 5 to 20 percent, and the Tm is 3+ The doping molar concentration of the ions is 0.5 to 2 percent; in the intermediate shell of the second upconversion nanoparticle, yb 3+ The doping molar concentration of the ions is 5 to 20 percent.
Specifically, the second upconversion nanoparticle is NaGdF 4 :Yb/Tm@NaNdF 4 :Yb@NaGdF 4 In the core of the second upconversion nanoparticle, yb 3+ Doping molarity of ion is 20%, tm 3+ The doping molar concentration of the ions is 1%; in the intermediate shell of the second upconversion nanoparticle, yb 3+ The doping molarity of the ions was 10%.
Specifically, the biomarker can be a serum tumor marker, such as human chorionic gonadotropin (hCG), alpha-fetoprotein (AFP), and the like.
In another embodiment, the first upconversion nanoparticle is NaYF 4 @NaYbF 4 :Er@NaYF 4 Said Er 3+ The doping molar concentration of the ions is 2 percent;
the second upconversion nanoparticle is NaGdF 4 :Yb/Tm@NaNdF 4 :Yb@NaGdF 4 Said Yb 3+ Doping molarity of ion is 20%, tm 3+ The doping molar concentration of the ions is 1 percent, and Yb is 3+ The doping molarity of the ions was 10%.
Specifically, the NaYF 4 @NaYbF 4 :2％Er@NaYF 4 The path of the medium luminous energy transfer is Yb 3+ →Er 3+ . But due to Er 3+ Can absorb 980nm and 808nm photons by itself, so the NaYF 4 @NaYbF 4 :2％Er@NaYF 4 Er of (2) 3+ Is low in concentration of Yb 3+ Is higher to inhibit the NaYF 4 @NaYbF 4 :2％Er@NaYF 4 Response at 808nm, therefore, the NaYF 4 @NaYbF 4 :2％Er@NaYF 4 The fluorescent material can emit fluorescence under the excitation of 980nm wavelength of near infrared light and hardly emit fluorescence under the excitation of 808nm wavelength of near infrared light.
Specifically, the NaGdF 4 :Yb(20/1％)/Tm@NaNdF 4 :10％Yb@NaGdF 4 Can have higher luminous intensity under the excitation of 808nm, and is doped with Nd absorbing photons of 808nm 3+ To realize the NaGdF 4 :Yb(20/1％)/Tm@NaNdF 4 :10％Yb@NaGdF 4 Absorption of 808nm photons, but due to Tm 3+ Energy transfer pathway Nd for luminescence 3+ →Yb 3+ →Tm 3+ Therefore Yb 3+ Is also necessary and takes into account if Yb 3+ The doping concentration is too high and when excited at 980nm, the compound will also respond to emit blue light. Taken together, the second upconversion nanoparticle doped Nd of the present application 3+ The ion concentration was 90%.
In a second aspect, the present application provides a method for preparing the carboxyl-modified first upconversion nanoparticle, comprising:
step 1, preparing first up-conversion nanoparticles;
step 2, performing hydrophilic carboxyl modification on the first upconversion nanoparticles to obtain carboxyl-modified first upconversion nanoparticles;
the preparation method of the carboxyl-modified second upconversion nanoparticle comprises the following steps:
step one, preparing second up-conversion nanoparticles;
and step two, performing hydrophilic carboxyl modification on the second upconversion nanoparticles to obtain carboxyl-modified second upconversion nanoparticles.
In another embodiment, the first upconversion nanoparticle is prepared by a coprecipitation method to prepare NaYF 4 The preparation method comprises the following steps of coating a nano-particle inner core of a matrix with Er sequentially on the surface of the inner core by adopting a thermal decomposition method 3+ Doped NaYbF 4 Intermediate shell of nanoparticles and NaYF of a matrix 4 A surface shell of the nanoparticle of (a);
the second upconversion nanoparticle preparation method is a coprecipitation method for preparing Yb-doped nanoparticles 3+ And Tm 3+ NaGdF of 4 The core of the substrate is then thermally decomposedThe surface of the inner core is coated with Yb 3+ Doped NaNdF 4 Intermediate shell of nanoparticles of a matrix and NaGdF 4 A surface shell layer of the nanoparticle of (1).
Specifically, the first upconversion nanoparticle can be prepared by adopting an existing conventional method; in step 1, the method for preparing the first upconversion nanoparticle comprises:
1. preparing NaYF by coprecipitation method 4 Nanoparticles of a matrix forming NaYF 4 A nanoparticle;
2. the NaYF is prepared by adopting a thermal decomposition method 4 The nano particles of the matrix are cores, and Er is coated outside the cores 3+ Doped NaYbF 4 Intermediate shell of nanoparticles of matrix to form NaYF 4 @NaYbF 4 Er nano-particles;
3. the NaYF is prepared by adopting a thermal decomposition method 4 The nano particles of the matrix are cores, and Er is sequentially coated outside the cores 3+ Doped NaYbF 4 Intermediate shell of nanoparticles of matrix and NaYF 4 Surface shell of nanoparticles of substrate constituting Yb 3 + And Er 3+ Codoped NaYF 4 @NaYbF 4 :Er@NaYF 4 。
More specifically, the method for preparing the first upconversion nanoparticle comprises the following steps:
1、NaYF 4 the synthesis method is a coprecipitation method. Taking oleic acid and 1-octadecene and a two-neck flask, adding an aqueous solution of yttrium acetate (III), heating to 150 ℃, and continuously reacting for 50min. And naturally cooling the system temperature to 45 ℃ and keeping the system temperature stable after the reaction is finished. A mixed methanol solution of ammonium fluoride and sodium hydroxide was added. After 1.5h the solution was warmed until the methanol was completely evaporated. Finally, introducing argon gas flow into the flask for protection, raising the reaction temperature to 280-310 ℃, reacting for 1h, and cooling to room temperature. Adding absolute ethanol to precipitate the nanoparticles, centrifuging at 6000-14000rpm for 3-20min until the nanoparticles are attached to the wall of the centrifuge tube, pouring out the supernatant, adding 4mL cyclohexane, and dispersing again.
2、NaYF 4 @NaYbF 4 Of ErThe synthesis method is a thermal decomposition method. Firstly, adding oleic acid and 1-octadecene into a double-neck flask, then adding ytterbium trifluoroacetate, erbium trifluoroacetate and sodium trifluoroacetate, heating to 110-130 ℃ under stirring until the solution is yellow, and vacuumizing for 30min to obtain NaYbF 4 Er shell precursor. In another two-necked flask, oleic acid and 1-octadecene were added, and NaYbF was added 4 Er precursor and NaYF prepared by the method 4 Heating the nano-particle solution to 110-130 ℃, maintaining for 1h, vacuumizing for 10-30min until no bubbles are generated during vacuumizing, and then rapidly heating to 280-310 ℃ under the protection of argon gas and maintaining for 1h. Cooling to room temperature, adding anhydrous ethanol to allow nanoparticles to aggregate, centrifuging at 6000-14000rpm for 3-20min, discarding the upper layer waste liquid, and dispersing the nanoparticles in cyclohexane.
3、NaYF 4 @NaYbF 4 :Er@NaYF 4 The synthesis method of (2) is a thermal decomposition method. Firstly, adding oleic acid and 1-octadecene into a double-neck flask, then adding yttrium trifluoroacetate and sodium trifluoroacetate, heating to 110-130 ℃ under stirring until the solution is yellow, and vacuumizing for 30min to obtain NaYF 4 And (5) a shell layer precursor. Adding oleic acid and 1-octadecene into another double-neck flask, and adding NaYF 4 Precursor and NaYF prepared by method 4 @NaYbF 4 Heating Er solution to 110-130 deg.c, maintaining for 45min, vacuumizing for 10-30min until no bubble is produced, and raising temperature to 280-310 deg.c under the protection of argon gas for 1 hr. Cooling to room temperature, adding anhydrous alcohol to precipitate the nanoparticles, centrifuging at 6000-14000rpm for 3-20min, discarding the upper layer waste liquid, and dispersing the nanoparticles in cyclohexane.
Specifically, in the step one, the second upconversion nanoparticles can be prepared by using an existing conventional method, and the second upconversion nanoparticles are prepared by using a coprecipitation method, that is, a method for synthesizing NaGdF by using a coprecipitation method, as the first upconversion nanoparticles 4 Yb/Tm, and then preparing NaGdF by adopting a thermal decomposition method 4 :Yb/Tm@NaNdF 4 Yb, and finally coating NaYF by thermal decomposition method 4 Obtaining NaGdF4: yb/Tm @ NaNdF 4 :Yb@NaGdF 4 。
Specifically, in step 2, the first upconversion nanoparticle may be modified with hydrophilic carboxyl group by using a conventional method to obtain a carboxyl group modified first upconversion nanoparticle, for example, by using a microemulsion method or a polyacrylic acid (PAA) coating method.
Specifically, the step 2 specifically includes: subjecting the first upconversion nanoparticles (NaYF) prepared in step 1 to a reaction 4 @NaYbF 4 :Er@NaYF 4 ) And (3) carrying out ultrasonic treatment on the solution for 15min, adding the nanoparticle solution into cyclohexane, adding lgepal CO-520 and TEOS, and violently stirring for 5-10min. Ammonia and deionized water were added and stirred overnight to form a microemulsion. Next, TEOS is added to the mixture and stirred for 180min, then the aqueous solution of carboxyethyl silanetriol sodium is added, the mixture is subjected to ultrasonic treatment for 10-15min, and the mixture is stirred for 45-90min. And (3) coagulating the obtained carboxyl modified first upconversion nanoparticles by using N, N-Dimethylformamide (DMF), centrifuging at 10000-12000rpm for 10-16min, discarding the supernatant, dispersing the precipitate by using isopropanol, centrifuging by the same method, discarding the supernatant, and dispersing the precipitate by using deionized water for later use.
Specifically, the concentration of the ammonia water in step 2 is 25wt.%, and the concentration of the aqueous solution of carboxyethyl silanetriol sodium is 25wt.%.
Specifically, in the second step, the second upconversion nanoparticle may be modified with hydrophilic carboxyl group by using a conventional method to obtain a carboxyl group modified second upconversion nanoparticle, for example, by using a microemulsion method or a polyacrylic acid (PAA) coating method.
Specifically, please refer to the method in step 2 for the hydrophilic carboxyl modification method of the second upconversion nanoparticle in step two.
In a third aspect, the present application provides a use of the set of up-converting nanoparticles or the set of up-converting nanoparticles prepared by the preparation method for detecting biomarkers.
Specifically, a first biomarker secondary antibody and a second biomarker secondary antibody are fixed on a microplate to obtain a microplate in which the second antibody is fixed on the microplate;
coupling a first biomarker first antibody to the carboxyl-modified first upconversion nanoparticle to obtain a first probe; coupling a second biomarker first antibody to the carboxyl-modified second upconversion nanoparticle to obtain a second probe;
adding a sample to be detected into the microporous plate, and removing the excessive sample to be detected; then, after the micro-porous plate is closed, incubating the first probe and the second probe on the micro-porous plate; washing the microplate, then collecting the fluorescence intensity of each hole in the microplate under 980nm excitation light and 808nm excitation light respectively, and calculating according to the standard curves of the first biomarker and the second biomarker to obtain the concentrations of the first biomarker and the second biomarker in the sample to be detected.
In another embodiment, the method for preparing the first probe includes: performing an EDC/NHS cross-linking method to react the first upconversion nanoparticles subjected to carboxyl functionalization treatment with amino groups on a first antibody to complete antibody coupling, so as to obtain first biomarker first antibodies coupled with the carboxyl modified first upconversion nanoparticles;
the preparation method of the second probe comprises the following steps: and (3) reacting the carboxyl functionalized second upconversion nanoparticles with the amino on the first antibody of the second biomarker by adopting an EDC/NHS crosslinking method to complete the coupling of the antibody, so as to obtain the carboxyl modified second upconversion nanoparticles coupled with the first antibody of the second biomarker.
Specifically, the first antibody is a first antibody of a first biomarker; the second antibody is a primary antibody for the second biomarker.
Specifically, the method of the first probe comprises the following steps:
the carboxyl-modified first upconversion nanoparticle solution synthesized by the above method was washed twice with 2-morpholinoethanesulfonic acid (MES) buffer, centrifuged, and dissolved in 0.01M MES solution. mu.L of a solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) dissolved at a concentration of 10mg/mL in MES buffer and 100. Mu.L of a solution of N-hydroxysuccinimide (NHS) dissolved at a concentration of 10mg/mL in MES buffer were added to the above solution of carboxyl-modified first upconversion nanoparticles, and shaken slowly at room temperature for 30min to allow the carboxyl groups to be activated by EDC/NHS coupling. The activated carboxyl modified primary upconverting nanoparticles were centrifuged for 15min at 14000rpm and washed twice with MES buffer. After discarding the supernatant, an antibody against the biomarker (e.g., monoclonal antibody mAb 1) dissolved in MES buffer was added to the activated first upconverting nanoparticle, and the solution was then allowed to stand at room temperature for 1h. To quench excess activated carboxyl sites, tris buffer was added to the reaction solution and incubated at room temperature for 15min. The first upconverting nanoparticles were then washed with Phosphate Buffered Saline (PBS) containing 0.1wt.% Bovine Serum Albumin (BSA) by a centrifugation step at 11000rpm for 12 min. Finally, the first upconverting nanoparticle conjugated to the anti-biomarker antibody is resuspended and stored in PBS.
Specifically, the method of the second probe is similar to the method described above.
Specifically, the application includes: the up-conversion nanoparticle group is used as an up-conversion fluorescence immunoadsorption analysis and detection biomarker; up-conversion fluorescent immunoadsorption assay is abbreviated as ULISA (upper-linked immunosorbent assay).
Specifically, the method for detecting the biomarker by using the upconversion nanoparticle group as the upconversion fluorescence immunoadsorption analysis comprises the following steps:
1) Preparing a second antibody buffer solution for anti-biomarker A and a second antibody buffer solution for anti-biomarker B;
2) Preparing an antibody against biomarker a coupled to a first carboxyl-modified upconverting nanoparticle (denoted as a first probe) and an antibody against biomarker B coupled to a second carboxyl-modified upconverting nanoparticle (denoted as a second probe);
3) Adding the two second antibody buffer solutions of the biological marker A and the biological marker B into a microplate simultaneously, washing the microplate by using a washing buffer solution to remove excessive antibodies, blocking by using a blocking buffer solution, and washing the microplate by using the washing buffer solution to remove the excessive blocking buffer solution;
4) Adding the biomarker A with the equal gradient concentration into the microporous plate, washing to remove the excessive biomarker A, adding the first probe and the second probe into the microporous plate at the same time, and incubating;
5) After the incubation is finished, washing the microplate, and then collecting the fluorescence intensity of the first probe combined with the biomarker A in the microplate under 980nm excitation light;
6) And fitting a four-parameter logic curve to a standard curve of the biomarker A, and drawing an error bar in parallel with three experiments. The form of the four parameter logistic regression curve is as follows:
Figure BDA0003712874030000081
in the above formula, A 1 Background fluorescence intensity as a function of theory; a. The 2 Theoretical maximum fluorescence intensity; x is biomarker concentration; x is the number of 0 When the biomarker theoretically binds 50% of the biomarker to the antibody; y is the fluorescence intensity; p is x 0 The slope of the curve.
The limit of detection (LOD) is calculated as follows, s 0 Is shown as A 1 Standard deviation of (d);
y(LOD)＝A 1 +3s 0
LOD＝y -1 (y(LOD))
7) The above procedure was repeated to obtain the standard curve and detection limit for biomarker B (except that the excitation laser was 808nm and an isocratic concentration of biomarker B was used).
8) Adding the second antibody buffer solution for the anti-biomarker A and the anti-biomarker B into a microplate, washing the microplate with a washing buffer solution to remove excess antibodies, blocking with a blocking buffer solution, and washing the microplate with the washing buffer solution to remove excess blocking buffer solution;
9) Adding a sample to be detected into the microporous plate, washing to remove excessive sample to be detected, adding the first probe and the second probe into the microporous plate, and incubating;
10 Washing the microplate after the incubation is finished, then collecting the fluorescence intensity of each hole in the microplate under 980nm excitation light and 808nm excitation light respectively, and calculating to obtain the concentrations of the biomarker A and the biomarker B in the sample to be detected according to the standard curves of the biomarker A and the biomarker B.
Further, when the anti-biomarker A is AFP and the biomarker B is hCG, the method for detecting the biomarker by using the upconversion nanoparticle group as the enzyme-labeled antigen or the enzyme-labeled antibody of ELISA comprises the following steps: as shown in FIG. 3, the first probe is NaYF modified with a first antibody against AFP 4 @NaYbF 4 :1％Er@NaYF 4 The second probe is NaGdF modified with hCG first antibody 4 :Yb(20/1％)/Tm@NaNdF 4 :10％Yb@NaGdF 4 Up-converting nanoparticles; firstly, attaching an AFP second antibody to a microplate, attaching an hCG second antibody to the microplate, then adding a sample to be detected to the microplate, wherein if the sample to be detected contains AFP and/or hCG antigen, the AFP antigen can be specifically combined with the AFP second antibody, and the hCG antigen and the hCG second antibody are specifically combined; then, incubating the microporous plate with a first probe and/or a second probe, wherein an AFP first antibody of the first probe is specifically combined with an AFP antigen, and an hCG first antibody of the second probe is specifically combined with an hCG antigen; then, under excitation light of 980nm and 808nm, the first probe emits fluorescence at about 541nm, the second probe emits fluorescence at about 476nm, spectrum files of two biomarkers are generated, and the software reads values to calculate; and finally, comparing the detected values in the sample to be detected according to a pre-drawn standard curve to obtain the concentration results of the AFP and hCG antigens in the sample to be detected.
Compared with the existing rare earth-based multi-biomarker detection technology, the material adopted in the application can realize the specific detection of dual excitation-dual emission-dual detection, meets the requirements of time cost control and operation process simplification, and has extremely high accuracy in the detection of each marker.
Compared with up-conversion based on quantum dots and organic molecules, the rare earth luminescent material has high luminescent stability, narrow signal peak in spectrum and small mutual interference of fluorescence. The fluorescent probe is fixed by a synthesis method, so that a large amount of probes can be produced and stored for use at one time;
compared with the technology using the test strip, the up-conversion nanoparticle group provided by the application can adopt a double-antibody sandwich experimental method based on a microporous plate, has higher specificity, and reduces the possibility of false positive;
compared with methods based on light absorption, such as an enzyme-linked immunosorbent assay, the fluorescent immunoadsorption method based on emitted light for the upconversion nanoparticle group provided by the application has higher sensitivity, and the possibility of false negative is reduced;
the upconversion nanoparticle group provided by the application has the advantages of small interference of upconversion luminescence excited by near infrared, high specificity of antigen-antibody combination and reduction of the possibility of false positive.
To sum up, the application provides an up-conversion nanoparticle group that binary channels detected biomarker makes breakthrough improvement to traditional enzyme-linked immunosorbent assay, and the application provides an up-conversion nanoparticle group that binary channels detected biomarker, can realize detecting biomarker under the condition that does not use the enzyme, and the detection signal of this application is the fluorescence emission signal based on near-infrared up-conversion excitation moreover, and background interference is little, sensitivity promotes to some extent, and the detection limit also improves to some extent. The application can carry out the same pretreatment and detection experiments on the sample, and then respectively read data, thereby achieving the purpose of simultaneous detection.
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In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
Fig. 1 is a component schematic diagram of an upconversion nanoparticle set for dual channel biomarker detection provided in an embodiment of the present application, where the upconversion nanoparticle set includes NaYF 4 @NaYbF 4 :1％Er@NaYF 4 And NaGdF 4 :Yb/Tm(20/1％)@NaNdF 4 :10％Yb；
FIG. 2 is a schematic diagram of a prior art enzyme-linked immunosorbent assay;
fig. 3 is a schematic diagram of biomarker detection implemented by a two-channel biomarker detection upconversion nanoparticle set provided in an embodiment of the present application;
fig. 4 is a fluorescence spectrum of the set of upconversion nanoparticles for dual-channel biomarker detection provided in an embodiment of the present application at a corresponding excitation wavelength, where NP1 excited at 980nm is a first upconversion nanoparticle and NP2 excited at 808nm is a second upconversion nanoparticle;
FIG. 5 is a standard curve of the two-channel detection biomarker upconverting nanoparticle set AFP provided in the present application, in which solid symbols represent coordinate points of a standard sample, open circles represent coordinates corresponding to detection limits, and error bars represent standard deviations of three parallel experimental data;
fig. 6 is a standard curve of hCG detection using the two-channel detection biomarker upconversion nanoparticle set provided in the embodiment of the present application, where solid symbols represent coordinate points of a standard sample, open circles represent coordinates corresponding to detection limits, and error bars represent standard deviations of data of three-time parallel experiments.
Detailed Description
The application provides an up-conversion nanoparticle group for detecting biomarkers through two channels and a preparation method and application thereof, and is used for solving the technical defects that in the prior art, ELISA detection stability is limited, color development is easily interfered, and detection time is long.
The technical solutions in the embodiments of the present application will be described clearly and completely below, and it is obvious that the described embodiments are only some embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The raw materials and reagents used in the following examples are commercially available or self-made.
The following is an explanation of the proper nouns appearing in the present application:
the rare earth referred to in the present application is scandium (Sc) element No. 21, yttrium (Y) element No. 39, and elements No. 57 to 71 (lanthanoid elements) in the periodic table.
Doping in this application means that part of the ions in the original crystal lattice is regularly replaced by another ion.
The near infrared referred to in this application is defined according to ASTM (American society for testing and materials testing), which is an electromagnetic wave in the range of 780 to 1100nm, and the near infrared laser used in this application is 808nm and 980 nm.
Fluorescence as referred to herein is photoluminescence, i.e., the process in which an electron of a substance transitions from a ground state absorbing a photon to an excited state, and returns to the ground state through a series of changes to emit a photon.
The upconversion material referred to in this application absorbs photons of low energy and emits photons of high energy.
The tumor markers referred to in the application are substances capable of reflecting tumor formation and change.
Antigens referred to in this application: any substance that can induce the immune system to produce antibodies, including viruses, pathogenic bacteria, pollen, etc., and the tumor markers AFP and hCG in this application are two antigens.
Antibodies referred to in the present application: immunoglobulins, a class of proteins secreted by plasma cells (effector B cells) for neutralizing antigens.
Example 1
The present embodiments provide for the synthesis of 980nm excited first up-conversion nanoparticles:
NaYF 4 the synthesis method is called coprecipitation method. 6mL of oleic acid and 14mL of 1-octadecene were taken out and a two-necked flask, and 4.0mL of a 0.2M aqueous solution of yttrium (III) acetate was added thereto, and the mixture was heated to 150 ℃ with stirring and allowed to react for 50 minutes. And naturally cooling the system temperature to 45 ℃ and keeping the system temperature stable after the reaction is finished. A mixed methanol solution of 7.2mL of 0.4M ammonium fluoride and 2mL of 1M sodium hydroxide was added. After 1.5h the solution was warmed until the methanol had evaporated completely. Finally, introducing argon gas flow into the flask for protection, raising the reaction temperature to 290 ℃, reacting for 1h, and cooling to room temperature. Adding anhydrous ethanol to allow nanoparticles to aggregate, centrifuging at 6000rpm for 3min, discarding the upper layer waste liquid, dispersing the nanoparticles in 4mL cyclohexane, and storing in a refrigerator at 4 deg.CAnd (4) storing.
NaYF 4 @NaYbF 4 2% Er was synthesized by thermal decomposition. Firstly, 10mL of oleic acid and 10mL of 1-octadecene are added into a two-neck flask, then 1.96mL of ytterbium trifluoroacetate, 0.2M ytterbium trifluoroacetate, 0.04mL of erbium trifluoroacetate, 2mL of sodium trifluoroacetate and 0.4M sodium trifluoroacetate are added, the mixture is heated to 125 ℃ under stirring and then is maintained for 1h, and the mixture is vacuumized for 30min to obtain NaYbF 4 2% of Er precursor. In another two-necked flask, 1mL oleic acid and 9mL 1-octadecene were added, and 10mL NaYbF was added 4 2% Er precursor and 2mL NaYF prepared by the above method 4 Heating the nano-particle solution to 110 ℃, maintaining for 30min, vacuumizing for 10min, and then rapidly heating to 290 ℃ under the protection of argon and maintaining for 40min. After cooling to room temperature, adding absolute ethyl alcohol to aggregate the nanoparticles, centrifuging at 6000rpm for 3min, discarding the upper layer waste liquid, dispersing the nanoparticles in 2mL cyclohexane, and storing in a refrigerator at 4 ℃.
NaYF 4 @NaYbF 4 :2％Er@NaYF 4 The synthesis method of (2) is a thermal decomposition method. Adding 10mL of oleic acid and 10mL of 1-octadecene into a two-necked flask, adding 4mL of 0.2M yttrium trifluoroacetate, 2mL of 0.4M sodium trifluoroacetate, heating to 125 ℃ under stirring, maintaining for 1h, and vacuumizing for 30min to obtain NaYF 4 And (3) precursor. In another two-necked flask, 1mL oleic acid and 9mL 1-octadecene were added, and 10mL NaYF was added 4 Precursor and 2mL NaYF prepared by the method 4 @NaYbF 4 2 percent Er nanoparticle solution is heated to 110 ℃ and then maintained for 30min, vacuumized for 10min, and then rapidly heated to 290 ℃ under the protection of argon and maintained for 40min. After cooling to room temperature, adding absolute ethyl alcohol to aggregate the nanoparticles, centrifuging at 6000rpm for 3min, discarding the upper layer waste liquid, dispersing the nanoparticles in 2mL cyclohexane, and storing in a refrigerator at 4 ℃.
Comparative example 1
The comparative examples of the present application provide different Er' s 3+ The fluorescent properties of the ion-doped up-conversion nanoparticles with molar concentration, the up-conversion nanoparticles with different core-shell structures and the up-conversion nanoparticles doped with different rare earth ions specifically comprise the following steps:
1. er doping was adjusted to a higher level by the method of example 1 3+ Ion(s)The NaYF is prepared according to the molar concentration of the NaYF 4 @NaYbF 4 :80％Er@NaYF 4 Upconverting the nanoparticles, measuring the fluorescence spectrum of the upconverting nanoparticles, and determining the NaYF from the fluorescence spectrum 4 @NaYbF 4 :80％Er@NaYF 4 The response of the upconverting nanoparticle under 808nm excitation is enhanced and it is not possible to use the second upconverting nanoparticle in conjunction with the determination of the biomarker.
2. Reference example 1 method without coating NaYF 4 Preparing NaYF on the surface shell layer of the nano particles of the matrix 4 @NaYbF 4 2% of Er Up-converted nanoparticles, the fluorescence spectrum of the Up-converted nanoparticles was measured, and the NaYF was found from the fluorescence spectrum 4 @NaYbF 4 2% the overall luminescence efficiency of the Er up-conversion nanoparticles was lower than that of NP1 of example 1, the fluorescence response at 980nm was smaller than that of NP1 of example 1, the luminescence effect was poor, and no response was observed at 808nm, and the biomarker could not be measured in combination with the second up-conversion nanoparticles.
3. Referring to the method of example 1, the intermediate shell of the first upconversion nanoparticle was doped Yb 3+ And Er 3+ Ion doped Yb 3+ The molar concentration of the ions is 8 percent, and Er is doped 3+ The molar concentration of the ions is 2 percent to prepare NaYF 4 @NaYF 4 :Yb/Er(8/2％)@NaYF 4 Upconverting the nanoparticles, measuring the fluorescence spectrum of the upconverting nanoparticles, and determining the NaYF from the fluorescence spectrum 4 @NaYF 4 :Yb/Er(8/2％)@NaYF 4 The response of the upconversion nanoparticles at 980nm was lower than that of NP1 of example 1, the luminescence effect was poor, there was almost no response at 808nm, and the biomarker could not be determined in combination with the second upconversion nanoparticles.
Example 2
The present embodiments provide for the synthesis of a 808nm excited second upconversion nanoparticle:
preparation of NaGdF by coprecipitation-thermal decomposition method 4 :Yb/Tm(20/1％)@NaNdF 4 :10％Yb@NaGdF 4 Namely, synthesis of NaGdF by coprecipitation 4 Yb/Tm (20/1%), then coating NaNdF by thermal decomposition method 4 10% of Yb and NaYF 4 Obtaining NaGdF 4 :Yb/Tm(20/1％)@NaNdF 4 :10％Yb@NaGdF 4 The method specifically comprises the following steps:
NaGdF 4 the synthesis of Yb/Tm (20/1%) is a coprecipitation method. A two-necked flask was charged with 8mL of oleic acid and 12mL of 1-octadecene, and 3.16mL of an aqueous solution of 0.2M gadolinium (III) acetate, 0.8mL of 0.2M ytterbium (III) acetate, and 0.4mL of 0.02M thulium (III) acetate, and the mixture was heated to 150 ℃ with stirring and reacted for 50min. And naturally cooling the system to 45 ℃ after the reaction is finished and keeping the temperature stable. 5.2mL of a mixed methanol solution of 0.4M ammonium fluoride and 2mL of 1M sodium hydroxide was added. After 1.5h the solution was warmed until the methanol was completely evaporated. Finally, introducing argon gas flow into the flask for protection, raising the reaction temperature to 290 ℃, reacting for 1h, and cooling to room temperature. Adding anhydrous ethanol to allow the nanoparticles to aggregate, centrifuging at 6000rpm for 3min, discarding the upper layer waste liquid, dispersing the nanoparticles in 4mL cyclohexane, and storing in a refrigerator at 4 deg.C.
NaGdF 4 :Yb/Tm(20/1％)@NaNdF 4 10% by weight of Yb. Firstly, 10mL of oleic acid and 10mL of 1-octadecene are added into a double-neck flask, then 1.8mL of neodymium trifluoroacetate, 0.2M of neodymium trifluoroacetate, 0.2mL of ytterbium trifluoroacetate, 2mL of ytterbium trifluoroacetate and 0.4M of sodium trifluoroacetate are added, the mixture is heated to 125 ℃ under stirring and then is maintained for 1 hour, and the mixture is vacuumized for 30 minutes to obtain NaNdF 4 10% of Yb precursor. In another two-necked flask, 1mL oleic acid and 9mL 1-octadecene were added, and 10mL NaNdF was added 4 10% of Yb precursor and 2mL of NaGdF prepared by the above method 4 1 percent of Tm nanoparticle solution, heating to 110 ℃, maintaining for 30min, vacuumizing for 10min, and then rapidly heating to 290 ℃ under the protection of argon and maintaining for 40min. After cooling to room temperature, adding absolute ethyl alcohol to aggregate the nanoparticles, centrifuging at 6000rpm for 3min, discarding the upper layer waste liquid, dispersing the nanoparticles in 2mL cyclohexane, and storing in a refrigerator at 4 ℃.
NaGdF 4 :Yb/Tm(20/1％)@NaNdF 4 :10％Yb@NaGdF 4 The synthesis of (2) is a thermal decomposition method. Adding 10mL of oleic acid and 10mL of 1-octadecene into a two-necked flask, adding 4mL of gadolinium trifluoroacetate, 0.2M of gadolinium trifluoroacetate, 2mL of sodium trifluoroacetate and 0.4M of sodium trifluoroacetate, heating to 125 ℃ under stirring, maintaining for 1h, and vacuumizing for 30min to obtain NaGdF 4 And (3) precursor. At the other double neckThe flask was charged with 1mL of oleic acid and 9mL of 1-octadecene, and 10mL of NaGdF was added 4 Precursor and 2mL of NaGdF prepared by the method 4 :1％Tm@NaNdF 4 10 percent of Yb nano-particle solution, heating to 110 ℃, maintaining for 30min, vacuumizing for 10min, and then rapidly heating to 290 ℃ under the protection of argon and maintaining for 40min. After cooling to room temperature, adding absolute ethyl alcohol to aggregate the nanoparticles, centrifuging at 6000rpm for 3min, discarding the upper layer waste liquid, dispersing the nanoparticles in 2mL cyclohexane, and storing in a refrigerator at 4 ℃.
The embodiment of the present application measures fluorescence spectra of the first upconversion nanoparticle and the second upconversion nanoparticle under respective excitation of 980nm excitation light and 808nm laser, and the results are shown in fig. 4, and fig. 4 shows that the upconversion nanoparticle group for detecting a biomarker through two channels provided by the present application can emit fluorescence of different colors under different excitation lights.
Comparative example 2
Comparative examples of the present application provide different Tm 3+ The fluorescent properties of the ion-doped up-conversion nanoparticles with molar concentration, the up-conversion nanoparticles with different core-shell structures and the up-conversion nanoparticles doped with different rare earth ions specifically comprise the following steps:
1. referring to the method of example 2, the cores of the upconversion nanoparticles were not doped with Yb 3+ Ion to obtain NaGdF 4 :1％Tm@NaNdF 4 :10％Yb@NaGdF 4 Upconverting the nanoparticles, measuring the fluorescence spectrum of the upconverted nanoparticles, and obtaining the NaGdF from the fluorescence spectrum 4 :1％Tm@NaNdF 4 :10％Yb@NaGdF 4 The upconversion nanoparticles only have Yb in the core and the middle shell 3+ Ions can be excited effectively, resulting in overall luminous efficiency less than NP2 of example 2, poor luminous efficacy, and inability to assay biomarkers with the first upconverting nanoparticles.
2. Referring to the method of example 2, the intermediate shell of the upconversion nanoparticles was not doped with Yb 3+ Ion to obtain NaGdF 4 :Yb/Tm(20/1％)@NaNdF 4 @NaGdF 4 Up-converting the nanoparticles, measuring the fluorescence spectrum of the up-converted nanoparticles, and determining the fluorescence spectrumNaGdF 4 :Yb/Tm(20/1％)@NaNdF 4 @NaGdF 4 The upconversion nanoparticles only have Yb in the core and the middle shell 3+ Ions can be effectively excited, resulting in overall luminous efficiency less than NP2 of example 2, poor luminous efficacy, and inability to assay biomarkers in combination with the first up-converting nanoparticles.
3. Referring to the method of example 2, the Yb increase in the core of the upconverting nanoparticle 3+ Doping concentration of ions (Yb doping in the intermediate layer of upconversion nanoparticles) 3+ Ion doping concentration of 50%) to obtain NaGdF 4 :Yb/Tm(20/1％)@NaNdF 4 :50％Yb@NaGdF 4 The fluorescence spectrum of the upconverting nanoparticle was measured, and from the fluorescence spectrum, the NaGdF was found 4 :Yb/Tm(20/1％)@NaNdF 4 :50％Yb@NaGdF 4 The response at 980nm increased and biomarkers could not be measured in combination with the first up-converting nanoparticle.
4. Referring to the method of example 2, the core of the upconverting nanoparticle was simultaneously doped with Yb 3+ 、Tm 3+ And Nd 3+ Ions, the molar concentration of doping is respectively 10%, 1% and 70%, and the NaNdF is not coated 4 10% of Yb to obtain NaGdF 4 :Yb/Tm/Nd(10/1/70％)@NaGdF 4 Upconversion nanoparticles, measurement of the fluorescence spectrum of the upconversion nanoparticles, and determination of the NaGdF from the fluorescence spectrum 4 :Yb/Tm/Nd(10/1/70％)@NaGdF 4 Nd in the core of an upconversion nanoparticle 3 + Poor radius matching degree with other rare earth ions, resulting in reduced uniformity of the shape and size of the nano-particles, and serious cross relaxation phenomenon leading to Tm 3+ The ion luminescence intensity is reduced and it is not possible to use the first up-converting nanoparticle in conjunction with the determination of biomarkers.
5. Referring to the method of example 2, the upconversion nanoparticles were not coated with NaGdF 4 Preparing NaGdF on the surface shell layer of the nano-particles of the substrate 4 :Yb/Tm(20/1％)@NaNdF 4 10% Yb upconversion nanoparticles, measuring the fluorescence spectrum of the upconversion nanoparticles, and from the fluorescence spectrum, the NaGdF 4 :Yb/Tm(20/1％)@NaNdF 4 10% of Yb upconversion sodiumThe rice particles had lower luminescence efficiency and poor luminescence effect compared to NP2 of example 2, and could not be used in combination with the first up-converting nanoparticles for biomarker determination.
Example 3
The embodiment of the present application provides that the first upconversion nanoparticle and the second upconversion nanoparticle are subjected to carboxyl modification, and specifically include:
the first upconversion nanoparticle solution prepared above was sonicated for 15min, an appropriate amount of this solution was taken and added to 10mL cyclohexane, then 0.9mL lgepal CO-520 and 50. Mu.L TEOS were added and stirred vigorously for 10min. Add 30 μ L, 25wt.% ammonia water, and 30 μ L deionized water and stir overnight to form a microemulsion. Next, 12.5 μ L TEOS was added to the mixture and stirred for 180min, then 25 μ L of 25wt.% aqueous carboxyethylsilanetriol sodium solution was added, sonicated for 15min, and stirred for an additional hour. And (3) coagulating the obtained carboxyl modified first upconversion nanoparticles by using N, N-Dimethylformamide (DMF), centrifuging at 11000rpm for 15min, discarding the supernatant, dispersing the precipitate by using 1mL of isopropanol, centrifuging by using the same method, discarding the supernatant, dispersing the precipitate by using 1mL of deionized water to obtain carboxyl modified first upconversion nanoparticles, and storing in a refrigerator at 4 ℃.
The second upconversion nanoparticle solution prepared above was sonicated for 15min, an appropriate amount of this solution was taken and added to 10mL cyclohexane, then 0.9mL lgepal CO-520 and 50. Mu.L TEOS were added and stirred vigorously for 10min. Add 30 μ L, 25wt.% ammonia and 30 μ L deionized water and stir overnight to form a microemulsion. Next, 12.5 μ ltos was added to the mixture and stirred for 180min, then 25 μ L of 25wt.% aqueous carboxyethylsilanetriol sodium solution was added, sonicated for 15min, and stirred for an additional hour. And (3) performing coagulation on the obtained carboxyl modified second upconversion nanoparticles by using N, N-Dimethylformamide (DMF), centrifuging at 11000rpm for 15min, discarding the supernatant, dispersing the precipitate by using 1mL of isopropanol, centrifuging by using the same method, discarding the supernatant, dispersing the precipitate by using 1mL of deionized water to obtain carboxyl modified second upconversion nanoparticles, and storing in a refrigerator at 4 ℃.
Example 4
The embodiment of the application provides that two monoclonal antibodies mAb1 (AFP antibody and hCG antibody) are respectively coupled to carboxyl-modified upconversion nanoparticles based on EDC/NHS coupling reaction, and two probes (probes) are prepared:
the first upconversion nanoparticle of this example was labeled with AFP antibody and the second upconversion nanoparticle was labeled with hCG antibody.
mu.L of each of the carboxylic acid-modified nanoparticle solutions synthesized as described above was washed twice with 2-morpholinoethanesulfonic acid (MES) buffer (25mM, pH 6), centrifuged, and dissolved in 400. Mu.L of 0.01M MES solution. mu.L of 10mg/mL 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) solution in MES buffer and 100. Mu.L of 10mg/mL N-hydroxysuccinimide (NHS) solution in MES buffer were added to 400. Mu.L of the above washed carboxyl-modified nanoparticles and shaken slowly at room temperature for 30min to activate the carboxyl groups by EDC/NHS coupling. The activated nanoparticles (activated NP1 and activated NP 2) were centrifuged at 14000rpm for 15min and washed twice with MES buffer (25mM, pH 6), respectively. After discarding the supernatant, monoclonal antibody mAb1 (i.e., 0.5mg/mL, 57.6. Mu.L of AFP antibody dissolved in MES buffer and 0.5mg/mL, 57.6. Mu.L of hCG antibody dissolved in MES buffer) was added to the activated nanoparticles (activated NP1 and activated NP 2), and the solution was allowed to stand at room temperature for 1h. To quench excess activated carboxyl sites, 0.5mL Tris buffer (50mM, pH 7.4) was added to the reaction solution and incubated at room temperature for 15min. The nanoparticles were then washed with Phosphate Buffered Saline (PBS) containing 0.1wt.% Bovine Serum Albumin (BSA) by a centrifugation step at 11000rpm for 12 min. Finally, the antibody-coupled nanoparticles were resuspended and stored in 0.4mL PBS, resulting in two antibody-coupled carboxy-modified upconverting nanoparticles, i.e., an AFP antibody-coupled carboxy-modified first upconverting nanoparticle (denoted as first probe) and an hCG antibody-coupled carboxy-modified second upconverting nanoparticle (denoted as second probe).
Example 5
This embodiment provides and draws the immunoadsorption detection standard curve and the limit of detection (LOD) of above-mentioned first probe and second probe, specifically includes:
respectively combining two kinds ofMonoclonal antibody mAb2 (AFP antibody and hCG antibody, respectively) was dissolved in coating buffer (pH =9.6, 50mM NaHCO 3 /Na 2 CO 3 Buffer) was prepared as a 1.5. Mu.g/mL solution. The mAb2 solution was injected into a high binding 96 well microplate at 50. Mu.L per well, respectively, and allowed to stand overnight in a refrigerator at 4 ℃. Then washed with washing buffer (50mM, naH) 2 PO 4 /Na 2 HPO 4 0.01% Tween 20, pH 7.4) the plates were washed at 200. Mu.L per well to remove excess mAb2, and 200. Mu.L per well of blocking buffer (50mM containing 1% bovine serum albumin, naH at pH 7.4) 2 PO 4 /Na 2 HPO 4 ) And (6) sealing. The plate was then washed with 200. Mu.L of wash buffer per well to remove excess BSA. A series of concentration gradients of tumor marker antigen (AFP and hCG, respectively) were added to each well at 50. Mu.L per well. The plate was allowed to stand at 37 ℃ for 1h. After 2 washes, the fluorescent probe was added to each well in 50. Mu.L per well, and reacted at 37 ℃ for another 60 minutes. Finally, after four washes, drying, the fluorescence intensity of each well was measured and collected in a fixed light path, with AFP reading the fluorescence intensity corresponding to 541nm and hCG reading the fluorescence intensity corresponding to 476 nm.
In this example, a four-parameter logical curve is used to fit a standard curve, and an error bar is drawn in parallel with three experiments. The form of the four parameter logistic regression curve is as follows:
Figure BDA0003712874030000171
in the above formula, A 1 Background fluorescence intensity as a theory; a. The 2 Theoretical maximum fluorescence intensity; x is biomarker concentration; x is the number of 0 Is the concentration of biomarker bound to antibody by 50% theoretically; y is the fluorescence intensity; p is x 0 The slope of the curve.
The limit of detection (LOD) is calculated as follows, s 0 Represents the standard deviation of A1;
y(LOD)＝A 1 +3s 0
LOD＝y -1 (y(LOD))。
the resulting standard curves for the two antigens (AFP and hCG) were plotted as shown in fig. 5 and 6, and the parameters, correlation coefficients, calibration correlation coefficients, and detection limits of the standard curves for the two antigens (AFP and hCG) are shown in table 1. The normal concentration range of human chorionic gonadotropin (hCG) is less than 0.01IU/mL, and the normal concentration range of alpha-fetoprotein (AFP) is less than 10ng/mL, and the data in Table 1 show that the up-conversion nanoparticle group for the dual-channel detection biomarker can stably and sensitively detect the content of AFP and hCG, and can accurately detect the content of AFP and hCG even if the concentration of AFP and hCG is lower.
TABLE 1
AFP hCG
A 1 1236.45041±44.24658 2692.1935±75.56251
A 2 15625.12638±219.1164 27132.91633±800.95979
x 0 13.10537±1.50435ng/mL 1.52943±357.90729IU/mL
p 0.55664±0.02062 0.46323±0.03035
R 2 (correlation coefficient) 0.99973 0.99887
Corrected correlation coefficient 0.99952 0.99802
Detection limit 2.94×10 -3 ng/mL 6.26×10 -5 IU/mL
To sum up, the up-conversion nanoparticle group for the two-channel detection biomarker provided by the application can emit two different visible light rare earth up-conversion nanoparticles under excitation of two different wavelengths of laser. The up-conversion nanoparticle group for detecting the biomarker by two channels respectively marks two specific antibodies, so that the technology of double excitation-double emission-double detection can be realized, the technical scheme for detecting corresponding antigens by using the method based on immunoadsorption is adopted, and the possibility of expanding from two channels to multiple channels is realized.
The foregoing is only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims (10)

1. A set of two-channel biomarker-detecting upconverting nanoparticles, comprising:
a carboxyl-modified first upconverting nanoparticle and a carboxyl-modified second upconverting nanoparticle;
the first upconversion nanoparticle is doped with NaYF 4 The nano particles of the matrix are cores, and Er is sequentially coated outside the cores 3+ Doped NaYbF 4 Intermediate shell of nanoparticles and NaYF of a matrix 4 Surface shell of nanoparticles of substrate constituting Yb 3 + And Er 3+ Codoped NaYF 4 @NaYbF 4 :Er@NaYF 4 ；
The second upconversion nanoparticle is doped with Yb 3+ And Tm 3+ With NaGdF 4 The nano particles of the matrix are cores, and Yb is coated outside the cores in sequence 3+ Doped NaNdF 4 Intermediate shell of nanoparticles of a matrix and NaGdF 4 Surface shell of nanoparticles of a matrix constituting Nd 3+ 、Yb 3+ And Tm 3+ Co-doped NaGdF 4 :Yb/Tm@NaNdF 4 :Yb@NaGdF 4 ；
The NaYF 4 @NaYbF 4 :Er@NaYF 4 The surface of (2) is modified with carboxyl; the NaGdF 4 :Yb/Tm@NaNdF 4 :Yb@NaGdF 4 The surface of (2) is modified with carboxyl.
2. The set of upconverting nanoparticles according to claim 1, wherein the first upconverting nanoparticle fluoresces a first color under excitation of near infrared 980nm wavelength; the second up-conversion nanoparticles emit fluorescence of a second color under the excitation of the wavelength of 808nm near infrared light.
3. The set of upconverting nanoparticles according to claim 1, wherein the Er is present in an intermediate shell of the first upconverting nanoparticle 3+ The doping molar concentration of the ions is 2 to 100 percent.
4. The set of upconverting nanoparticles of claim 1, wherein the Yb is in the core of the second upconverting nanoparticle 3+ The doping molar concentration of ions is 5% -20%, and the Tm is 3+ Doping molarity of ionsThe degree is 0.5% -2%; in the intermediate shell of the second upconversion nanoparticle, yb 3+ The doping molar concentration of the ions is 5 to 20 percent.
5. The set of upconversion nanoparticles according to claim 1, wherein the first upconversion nanoparticle is a NaYF 4 @NaYbF 4 :Er@NaYF 4 Said Er 3+ The doping molar concentration of the ions is 2 percent;
the second upconversion nanoparticle is NaGdF 4 :Yb/Tm@NaNdF 4 :Yb@NaGdF 4 In the core of the second upconversion nanoparticle, yb 3+ Doping molarity of ion is 20%, tm 3+ The doping molar concentration of the ions is 1 percent;
in the intermediate shell of the second upconversion nanoparticle, yb 3+ The doping molarity of the ions was 10%.
6. The method of preparing a set of upconverting nanoparticles according to claim 1, wherein the method of preparing the first carboxyl-modified upconverting nanoparticle comprises:
step 1, preparing first up-conversion nanoparticles;
step 2, performing hydrophilic carboxyl modification on the first upconversion nanoparticles based on a microemulsion method to obtain carboxyl-modified first upconversion nanoparticles;
the preparation method of the carboxyl-modified second upconversion nanoparticle comprises the following steps:
step one, preparing second up-conversion nanoparticles;
and step two, carrying out hydrophilic carboxyl modification on the second upconversion nanoparticles based on a microemulsion method to obtain carboxyl-modified second upconversion nanoparticles.
7. The preparation method of claim 6, wherein the first upconversion nanoparticle is prepared by coprecipitation method to prepare NaYF 4 Nanoparticle cores of the matrix, followed by application of heatThe decomposition method is to coat Er on the surface of the inner core in sequence 3+ Doped NaYbF 4 Intermediate shell of nanoparticles and NaYF of a matrix 4 A surface shell of nanoparticles of a substrate;
the second upconversion nanoparticle preparation method is a coprecipitation method for preparing Yb-doped nanoparticles 3+ And Tm 3+ NaGdF of 4 The nano-particle core of the substrate is coated with Yb on the surface of the core in sequence by adopting a thermal decomposition method 3+ Doped NaNdF 4 Intermediate shell of nanoparticles of a matrix and NaGdF 4 A surface shell of nanoparticles of a matrix.
8. Use of the set of upconverting nanoparticles according to any one of claims 1 to 5 or the set of upconverting nanoparticles produced by the method of preparation according to claim 6 or 7 for the detection of biomarkers.
9. The application according to claim 8, wherein the application comprises:
fixing a first biomarker secondary antibody and a second biomarker secondary antibody on a microplate to obtain a microplate on which the second antibody is fixed;
coupling a first biomarker first antibody to the carboxyl-modified first upconversion nanoparticle to obtain a first probe; coupling a second biomarker first antibody to the carboxyl-modified second upconversion nanoparticle to obtain a second probe;
adding a sample to be detected into the microporous plate, and removing the excessive sample to be detected; then, after the microporous plate is closed, incubating the first probe and the second probe on the microporous plate; washing the microplate, then collecting the fluorescence intensity of each hole in the microplate under 980nm excitation light and 808nm excitation light respectively, and calculating according to the standard curves of the first biomarker and the second biomarker to obtain the concentrations of the first biomarker and the second biomarker in the sample to be detected.
10. The use of claim 9, wherein the first probe is prepared by a method comprising: performing an EDC/NHS cross-linking method to react the first carboxyl-functionalized upconversion nanoparticles with amino groups on a first antibody to complete antibody coupling, so as to obtain first biomarker first antibody coupled with the carboxyl-modified first upconversion nanoparticles;
the preparation method of the second probe comprises the following steps: and (3) reacting the second upconversion nanoparticles subjected to carboxyl functionalization treatment with amino groups on the first antibody of the second biomarker by adopting an EDC/NHS crosslinking method to complete antibody coupling, so as to obtain the second upconversion nanoparticles subjected to carboxyl modification by coupling of the first antibody of the second biomarker.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117089350A (en) * 2023-07-21 2023-11-21 中国科学院福建物质结构研究所 High-light-efficiency rare earth nano fluorescent material, preparation method thereof and application thereof in contrast agent

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102140344A (en) * 2010-02-03 2011-08-03 中国科学院福建物质结构研究所 Two-mode nanometer fluorescence labelling material based on rare earth doped sodium gadolinium fluoride core-shell structure and preparation method thereof
CN105441080A (en) * 2015-11-17 2016-03-30 中山大学 Method for quickly preparing NaYF:Yb/Er upconversion nano particles having different phases
CN107286924A (en) * 2017-06-13 2017-10-24 复旦大学 Upper conversion nano crystalline material of the infrared ray excited orthogonal fluorescent emission of redgreenblue and preparation method thereof
CN107828408A (en) * 2017-10-12 2018-03-23 复旦大学 The lower conversion nano fluorescence probe of the window of near-infrared second transmitting and its synthetic method
CN111303878A (en) * 2019-04-15 2020-06-19 上海大学 Up-conversion luminescent nanoparticle preparation and chromatography test strip based on double excitation and double emission and detection method
US20210139772A1 (en) * 2018-07-03 2021-05-13 Rutgers, The State University Of New Jersey Luminescent layered composition and a method for using the composition
CN112903649A (en) * 2021-01-26 2021-06-04 上海大学 Double-excitation orthogonal emission up-conversion luminescence nanoparticle, multi-flux detection immunochromatography test paper and application thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102140344A (en) * 2010-02-03 2011-08-03 中国科学院福建物质结构研究所 Two-mode nanometer fluorescence labelling material based on rare earth doped sodium gadolinium fluoride core-shell structure and preparation method thereof
CN105441080A (en) * 2015-11-17 2016-03-30 中山大学 Method for quickly preparing NaYF:Yb/Er upconversion nano particles having different phases
CN107286924A (en) * 2017-06-13 2017-10-24 复旦大学 Upper conversion nano crystalline material of the infrared ray excited orthogonal fluorescent emission of redgreenblue and preparation method thereof
CN107828408A (en) * 2017-10-12 2018-03-23 复旦大学 The lower conversion nano fluorescence probe of the window of near-infrared second transmitting and its synthetic method
US20210139772A1 (en) * 2018-07-03 2021-05-13 Rutgers, The State University Of New Jersey Luminescent layered composition and a method for using the composition
CN111303878A (en) * 2019-04-15 2020-06-19 上海大学 Up-conversion luminescent nanoparticle preparation and chromatography test strip based on double excitation and double emission and detection method
CN112903649A (en) * 2021-01-26 2021-06-04 上海大学 Double-excitation orthogonal emission up-conversion luminescence nanoparticle, multi-flux detection immunochromatography test paper and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117089350A (en) * 2023-07-21 2023-11-21 中国科学院福建物质结构研究所 High-light-efficiency rare earth nano fluorescent material, preparation method thereof and application thereof in contrast agent

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