CN114137217A - FRET-based homogeneous immunoassay method and detection composition - Google Patents
FRET-based homogeneous immunoassay method and detection composition Download PDFInfo
- Publication number
- CN114137217A CN114137217A CN202111443663.7A CN202111443663A CN114137217A CN 114137217 A CN114137217 A CN 114137217A CN 202111443663 A CN202111443663 A CN 202111443663A CN 114137217 A CN114137217 A CN 114137217A
- Authority
- CN
- China
- Prior art keywords
- marked
- bismuth
- antibody
- antigen
- rare earth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Nanotechnology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to a FRET-based homogeneous immunoassay method and a detection composition, belonging to the technical field of analytical chemistry. Solves the technical problems of low sensitivity, high background fluorescence and the like of the existing FRET-based homogeneous immunoassay method. The detection composition comprises a bismuth-based material marked with an antigen and a rare earth doped luminescent nanocrystal marked with an antibody; or, the bismuth-based material marked with the antibody and the rare earth doped luminescent nanocrystal marked with the antigen are included; wherein, the antigen and the antibody are an antigen-antibody pair which can generate specific binding reaction; the bismuth-based material is a bismuth-based two-dimensional nano sheet material or a metal ion-doped bismuth-based two-dimensional nano sheet material. The invention also provides a method for detecting an antibody or an antigen by using the detection composition. The FRET-based homogeneous immunoassay method has the advantages of small background fluorescence interference, high detection sensitivity, low cost, simplicity, convenience in operation and the like.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a FRET-based homogeneous immunoassay method and a detection composition.
Background
Homogeneous immunoassay based on Fluorescence Resonance Energy Transfer (FRET) is a hotspot of research and application in recent years. FRET refers to a process of non-radiative energy transfer between a donor and an acceptor that occurs within a certain distance (typically <10 nm). The space-limited domain effect endows the FRET homogeneous detection technology with various advantages, such as no need of separation and purification of the object to be detected, simple operation method, high detection speed, good selectivity, wide application range, capability of simultaneously carrying out multi-component detection and the like. It follows that the choice of donor and acceptor is crucial to achieving the rapid, high assay sensitivity detection method described above. For donors, rare earth doped luminescent nanocrystal (RENCs) -based donor materials are the leading research edge in the field of analytical detection. RENCs are photon conversion luminescent materials which emit light from ultraviolet, visible to near infrared bands when excited by near infrared light (for example, 980, 800 or 1530nm), and have the characteristics of multi-band emission, narrow emission line, stable photochemical property, long fluorescence life, large (anti-) Stokes shift, no light bleaching phenomenon and adjustable spectrum. Compared with traditional donor materials such as organic dyes and quantum dots, the donor materials can effectively avoid biological autofluorescence and reduce scattering interference, so that RENCs become ideal donor materials for constructing sensing models in complex biological matrixes (blood, urine, saliva, organisms and the like).
Although RENC-FRET-based bio-detection techniques have attracted considerable attention in recent years, they have been far from practical use due to their low detection sensitivity. As can be seen from the energy transfer formula, the improvement of the energy transfer efficiency is to shorten the interaction distance between donor and acceptor, and to improve the light emitting efficiency of the donor. However, since RENCs are composed of rare earth ions with independent luminescence centers, and the energy transfer efficiency is inversely proportional to the sixth power of distance, only the luminescence center located at the surface interface and close enough to the acceptor can generate FRET with the acceptor, and the internal luminescence ion does not contribute to the process, as in the existing co-doped system (Yb)3+，Er3+) Surface-only Er3+Participate in energy transfer and thus severely reduce FRET efficiency. On the other hand, although the luminescence brightness of RENCs can be increased by suppressing the surface quenching effect by increasing the shell thickness of RENCs, the interaction distance between donor and acceptor is also increased, which leads to a limited improvement in detection sensitivity. Therefore, in order to overcome the above problems, a new design idea and a new method are developed, which are especially critical for developing a detection application with a wider application range and constructing a high-efficiency RENC-FRET detection method with high detection sensitivity and low fluorescence background.
Disclosure of Invention
In view of the above, the present invention provides a FRET-based homogeneous immunoassay method and a FRET-based homogeneous immunoassay composition, so as to solve the technical problems of low sensitivity, high background fluorescence, and the like of the conventional FRET-based homogeneous immunoassay method.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the present invention provides a test composition for detecting,
comprises a bismuth-based material marked with an antigen and a rare earth doped luminescent nanocrystal marked with an antibody;
alternatively, the first and second electrodes may be,
comprises a bismuth-based material marked with an antibody and a rare earth doped luminescent nanocrystal marked with an antigen;
the antigen and the antibody are an antigen-antibody pair which can generate specific binding reaction;
the bismuth-based material is a bismuth-based two-dimensional nano sheet material or a metal ion-doped bismuth-based two-dimensional nano sheet material.
It is preferable that the first and second liquid crystal layers are formed of,
the molar ratio of the bismuth-based material marked with the antigen to the rare earth doped luminescent nanocrystal marked with the antibody is 1 (1-10);
the molar ratio of the bismuth-based material marked with the antibody to the rare earth-doped luminescent nanocrystal marked with the antigen is 1 (1-10).
It is more preferable that the content of the organic compound,
the molar ratio of the bismuth-based material marked with the antigen to the rare earth doped luminescent nanocrystal marked with the antibody is 1: 1;
the molar ratio of the bismuth-based material marked with the antibody to the rare earth doped luminescent nanocrystal marked with the antigen is 1: 1.
Preferably, the bismuth-based two-dimensional nano flaky material is Bi2S3、Bi2Se3Or Bi2Te3。
It is preferable that the first and second liquid crystal layers are formed of,
the preparation method of the bismuth-based material marked with the antigen comprises the following steps:
mixing the bismuth-based material with a solution containing the antigen overnight, centrifuging, and dispersing the obtained precipitate in a buffer solution to obtain the solution of the bismuth-based material marked with the antigen.
The preparation method of the bismuth-based material marked with the antibody comprises the following steps:
mixing the bismuth-based material with the solution containing the antibody overnight, centrifuging, and dispersing the obtained precipitate in a buffer solution to obtain the solution of the bismuth-based material marked with the antibody.
It is preferable that the first and second liquid crystal layers are formed of,
the preparation method of the rare earth doped luminescent nanocrystal marked with the antibody comprises the following steps:
coating a carboxyl polymer on the rare earth doped luminescent nanocrystal, then mixing the coated rare earth doped luminescent nanocrystal with an activator and a solution containing an antibody, reacting, centrifuging, and dispersing the obtained precipitate in a buffer solution to obtain a solution of the rare earth doped luminescent nanocrystal marked with the antibody;
the preparation method of the rare earth doped luminescent nanocrystal marked with the antigen comprises the following steps:
and (2) coating the rare earth doped luminescent nanocrystal with a carboxyl polymer, then mixing the coated rare earth doped luminescent nanocrystal with an activator and a solution containing an antigen, reacting, centrifuging, and dispersing the obtained precipitate in a buffer solution to obtain the solution of the rare earth doped luminescent nanocrystal marked with the antigen.
The invention also provides a method for detecting the antibody by using the antigen detection composition, which comprises the following steps:
1) uniformly mixing the bismuth-based material solution marked with the antigen and the rare earth-doped luminescent nanocrystal solution marked with the antibody to obtain a detection system;
2) and adding a sample to be detected into the detection system, and performing emission spectrum detection.
The invention also provides a method for detecting antigen by using the antibody detection composition, which comprises the following steps:
1) uniformly mixing the bismuth-based material solution marked with the antibody and the rare earth-doped luminescent nanocrystal solution marked with the antigen to obtain a detection system;
2) and adding a sample to be detected into the detection system, and performing emission spectrum detection.
Preferably, the excitation light of the emission spectrum is near-infrared light of 700-1700 nm, and the emission light is visible light of 400-800 nm or near-infrared light of 900-1700 nm.
More preferably, the excitation light of the emission spectrum is near-infrared light of 800nm, and the emission light is visible light of 540nm, visible light of 650nm, near-infrared light of 1064nm, or near-infrared light of 1530 nm.
More preferably, the excitation light of the emission spectrum is near-infrared light of 980nm, and the emission light is visible light of 690nm, visible light of 800nm, near-infrared light of 1064nm, near-infrared light of 1180nm, or near-infrared light of 1530 nm.
More preferably, the excitation light of the emission spectrum is 1530nm near-infrared light, and the emission light is 650nm visible light, 800nm visible light, or 1180nm near-infrared light.
Compared with the prior art, the invention has the beneficial effects that:
the FRET-based homogeneous immunoassay method has the advantages of small background fluorescence interference, high detection sensitivity, low cost, simple method, convenient operation and the like.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings used in the detailed description will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive labor.
FIG. 1 shows a bismuth-based material (Bi) prepared in example 1 of the present invention2Se3Nanomaterial) scanning electron microscopy;
FIG. 2 shows a bismuth-based material (Bi) prepared in example 1 of the present invention2Se3Nanomaterial) absorption spectrum in the visible band;
FIG. 3 is a scanning electron micrograph of RENCs prepared in example 1 of the present invention;
FIG. 4 is an up-conversion emission spectrum of antibody-labeled RENCs prepared in example 1 of the present invention under excitation of 980nm light;
FIG. 5 shows a bismuth-based material (Bi) containing an antigen modification of antibody-labeled RENCs prepared in example 1 of the present invention2Se3Nanomaterial) the change in luminescence intensity of the upconverted nanomaterial;
FIG. 6 is a graph showing the change of luminescence intensity of the up-conversion nanomaterial after performing an immune competition reaction by introducing free antigen in example 1 of the present invention.
FIG. 7 is a scanning electron micrograph of RENCs prepared in example 2 of the present invention.
FIG. 8 is an up-conversion emission spectrum of antibody-labeled RENCs prepared in example 2 of the present invention under excitation with 980nm light.
Detailed Description
For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the detailed description, but it is to be understood that the description is intended to further illustrate the features and advantages of the invention and not to limit the claims to the invention.
The invention provides a detection composition, which comprises a bismuth-based material marked with an antigen and a rare earth doped luminescent nanocrystal marked with an antibody; or, the bismuth-based material marked with the antibody and the rare earth doped luminescent nanocrystal marked with the antigen are included; wherein, the antigen and the antibody are an antigen-antibody pair which can generate specific binding reaction.
In the present invention, the antigen and the antibody are not particularly limited, and theoretically, any pair of antigen and antibody capable of generating a specific binding reaction may be used, for example, the antigen may be neocorona antigen, PSA, or the like, and the antibody may be neocorona antibody IgM/IgG, or the like.
In the invention, the bismuth-based material is a bismuth-based two-dimensional nano sheet material or a metal ion-doped bismuth-based two-dimensional nano sheet material. The materials are known in the art and can be obtained by means well known to those skilled in the art, such as commercial or laboratory synthesis. For example, a commonly used bismuth-based two-dimensional nano-platelet material is Bi2S3、Bi2Se3Or Bi2Te3The invention preferably selects the bismuth-based two-dimensional nano flaky material Bi2Se3Or Bi2Te3. The doped metal ions can be rare earth ions, transition metal ions and the like, and the doping proportion is not limited. The invention provides a preparation method of a bismuth-based material marked with an antigen, which is not limited to the preparation method and specifically comprises the following steps: stirring and mixing the bismuth-based material and a solution containing the antigen for more than 12h, centrifuging, and dispersing the obtained precipitate in a buffer solution to obtain the bismuth-based material marked with the antigen, wherein the mass ratio of the bismuth-based material to the antigen is 1 (0.1-0.5), preferably 1: 0.2. The buffer is preferably Tris-hcl buffer at pH 7.2 and a concentration of 0.05M. The invention provides a preparation method of a bismuth-based material marked with an antibody, which is not limited to the preparation method and specifically comprises the following steps: stirring and mixing the bismuth-based material and the solution containing the antibody for more than 12 hours, centrifuging, and dispersing the obtained precipitate in a buffer solution to obtain the bismuth-based material marked with the antibody. The buffer is preferably Tris-hcl buffer at pH 7.2 and a concentration of 0.05M. In the present invention, the mass ratio of the bismuth-based material to the antibody is 1 (0.1 to 0.5), preferably 1: 0.2.
In the invention, the rare earth doped luminescent nanocrystal is the prior art and can be obtained by means well known by those skilled in the art such as commercial or laboratory synthesis. The rare earth doped luminescent nanocrystal can be a luminescent nanocrystal doped with a rare earth element, and the luminescent central ion is any rare earth element except Y, Lu and Gd, such as NaErF4@NaYF4. The luminescent nano-crystal can also be doped by a co-doping system of two or more rare earth ions, wherein the doping system can be Yb and Er, Yb and Tm, Yb and Nd, Yb and Ho and the like, such as NaErF4: YbEr. The core-shell structure is also possible.The invention provides a preparation method of a rare earth doped luminescent nanocrystal marked with an antibody, which is not limited to the preparation method and specifically comprises the following steps: dissolving the rare earth doped luminescent nanocrystal in a solvent (cyclohexane), precipitating with ethanol, centrifuging, dissolving in chloroform, adding a carboxyl polymer, fully mixing uniformly, performing rotary evaporation to remove the solvent, dissolving in deionized water, mixing with an activator and a solution containing an antibody or an antigen, reacting, centrifuging, and dispersing the obtained precipitate in a buffer solution to obtain the solution of the rare earth doped luminescent nanocrystal marked with the antibody or the antigen. The carboxyl polymer is preferably polymethacrylic acid grafted with octadecylamine, the grafting method is an EDC coupling method, and the modification proportion is 30-40%. The mass ratio of the rare earth doped luminescent nanocrystal to the carboxyl polymer is 1 (1-4). The activator is preferably EDC, or a mixture of EDC and NHS, and the antigen or antibody reacts with the activator in a ratio of 1: (1-5) equivalent. The mass ratio of the rare earth doped luminescent nanocrystal to the antigen or antibody is 1: (0.2 to 0.6), and more preferably 0.4. The reaction time is preferably 3 h. The reaction method is an EDC coupling method. The buffer is preferably Tris-HCl buffer.
In the invention, the molar ratio of the bismuth-based material marked with the antigen to the rare earth doped luminescent nanocrystal marked with the antibody is 1 (1-10), and the preferred molar ratio is 1: 1.
In the invention, the molar ratio of the bismuth-based material marked with the antibody to the rare earth doped luminescent nanocrystal marked with the antibody is 1 (1-10), and the preferred molar ratio is 1: 1.
The detection composition of the invention can be used for an antigen or antibody detection method, and comprises the following steps:
1) uniformly mixing the bismuth-based material solution marked with the antigen and the rare earth-doped luminescent nanocrystal solution marked with the antibody to obtain a detection system;
2) adding a sample to be detected into a detection system, and carrying out emission spectrum detection.
Alternatively, the first and second electrodes may be,
1) uniformly mixing the bismuth-based material solution marked with the antibody and the rare earth-doped luminescent nanocrystal solution marked with the antigen to obtain a detection system;
2) adding a sample to be detected into a detection system, and carrying out emission spectrum detection.
In the present invention, the sample to be detected is an antigen or an antibody.
In the invention, the exciting light of the emission spectrum is near infrared light of 700-1700 nm, and the emitted light is visible light of 400-800 nm or 900-1700 nm. Preferably, the excitation light of the emission spectrum is near-infrared light of 800nm, and the emission light is visible light of 540nm, visible light of 650nm, near-infrared light of 1064nm, or near-infrared light of 1530 nm; alternatively, the excitation light of the emission spectrum is near-infrared light of 980nm, and the emission light is visible light of 690nm, visible light of 800nm, near-infrared light of 1064nm, near-infrared light of 1180nm, or near-infrared light of 1530 nm; the emission spectrum of the excitation light was 1530nm, and the emission light was 650nm visible light, 800nm visible light, or 1180nm near-infrared light.
The invention also discloses a process parameter of the antigen/antibody detection method. Those skilled in the art can make appropriate modifications in view of the disclosure herein.
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail with reference to the following embodiments.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.
Example 1
1) Bismuth-based materials (Bi)2Se3Nanomaterial) preparation
0.372mmolBi (NO)3)3·.3H2O、0.560mmol NaSeO3And 0.4g PVP was placed in a 50mL three necked flask and 24mL ethylene glycol was added. Then putting the three-necked bottle into a magnetic stirring heater, introducing nitrogen for protection, heating to 60 deg.C, maintaining at the temperature for 30min, and dissolving the solid completely to obtain clear and transparent solution. Then heating the clear and transparent solution to 180 ℃, injecting a mixed solution of 0.8mL hydrazine hydrate and 8mL ethylene glycol at the temperature, reacting for 30min, reacting under the protection of nitrogen, refluxing by adopting cooling water, cooling to room temperature after the reaction is finished, and centrifugally purifying by using acetone once to obtain Bi2Se3Nano material, Bi obtained2Se3The nanomaterial was dispersed into 16mL of deionized water for use.
Bi obtained by using a scanning electron microscope and absorption spectrum detection2Se3Nanomaterial, results are shown in fig. 1 and 2. FIG. 1 shows Bi2Se3Scanning electron microscope images of the nano materials; FIG. 2 shows Bi2Se3The absorption spectrogram of the nano material in a visible light wave band can be seen from fig. 1 and 2, and the obtained Bi2Se3The nano material is uniform and has the size of about 100nm, and the absorption peak is located at 482 nm.
2) Preparation of RENCs
1mmol of rare earth chloride (including 0.78mmol YCl)3·6H2O，0.20mmol YbCl3·6H2O and 0.02mmol ErCl3·6H2O) was placed in a three-necked flask having a capacity of 50mL, and 15mL of octadecene and 6mL of oleic acid were added. And then putting the three-necked bottle into a magnetic stirring heater, introducing nitrogen for protection, heating to 160 ℃, maintaining the temperature for 30min, and obtaining a clear and transparent solution with light yellow color after chloride is completely dissolved. Then cooled to room temperature, 4mmol of NH will dissolve4F. Dropwise adding 2.5mmol of NaOH in 10mL of methanol solution into a three-necked bottle, heating to 70 ℃, keeping for 30min, completely evaporating the methanol in the solution, heating to 300 ℃, reacting for 90min, keeping introducing argon gas for protection in the reaction process, refluxing by using cooling water, cooling to room temperature after the reaction is finished, adding ethanol, centrifugally purifying for three times to obtain RENCs, and dissolving the RENCs into 4mL of cyclohexane for later use.
The obtained material was examined by scanning electron microscopy, and the results are shown in FIG. 3. FIG. 3 is a scanning electron micrograph of RENCs, and it can be seen from FIG. 3 that RENCs are obtained as uniform particles having a particle size of about 20 nm.
3) Preparation of antibody-labeled RENCs
Taking 1mL of RENCs cyclohexane solution, precipitating with ethanol, centrifuging, redissolving with chloroform, adding 50mg of carboxyl polymer (grafted octadecylamine polymethacrylic acid, the grafting method is an EDC coupling method, the modification ratio is 35 percent), fully mixing uniformly, removing the solvent by rotary evaporation, and dissolving with 2mL of deionized water for later use. Then, 0.1mL of the obtained aqueous material solution was centrifuged to obtain an upconverting nanomaterial, which was dispersed with 1mL of tris-HCl [ pH 7.2, 0.05M ], 8mg of nhs and 5mg of edc were added thereto, and after stirring them thoroughly and uniformly, 5mg of edc and 0.4mg of goat anti-bovine serum albumin were added thereto, and after stirring for 3 hours, the mixture was centrifuged to obtain RENCs labeled with an antibody, which was dissolved with 2mL of tris-HCl [ pH 7.2, 0.05M ] for use.
The emission spectrum of the obtained antibody-labeled RENCs with 980nm excitation light was detected, and the emission spectrum in the wavelength range of 450 to 750nm was measured as shown in FIG. 4. FIG. 4 is an up-conversion emission spectrum of antibody-labeled RENCs prepared in example 1 of the present invention under excitation with 980nm light. As can be seen from FIG. 4, the upconversion luminescence generated by RENCs under 980nm light excitation can be well matched with Bi2Se3The absorption matching can effectively realize RENCs and Bi2Se3And further detecting corresponding biological molecules according to the change of fluorescence signals of RENCs.
4) Bismuth-based material labeled with antigen
The bismuth-based material and a solution containing the antigen (the amount ratio of the bismuth-based material to the ovine IgG is 1:0.2) were mixed overnight, and then centrifuged, and the obtained precipitate was dispersed in 1mL of Tris-HCl [ pH 7.2, 0.05M ], to obtain a bismuth-based material labeled with the antigen.
5) Bismuth-based material with antibody-labeled RENCs introduced with antigen modification
Uniformly mixing the bismuth-based material solution marked with the antigen obtained in the step 4) and the rare earth-doped luminescent nanocrystal solution marked with the antibody obtained in the step 3) according to the molar ratio of the bismuth-based material marked with the antigen to the rare earth-doped luminescent nanocrystal marked with the antibody of 1:1 to obtain a detection system, adding an antigen sample to be detected (sheep IgG, 40 mu g/mL) into the detection system, and performing emission spectrum detection, wherein the result is shown in FIG. 5.
FIG. 5 is the change of luminescence intensity of up-conversion nanomaterials with antibody-labeled RENCs after the introduction of the antigen-modified bismuth-based material in example 1 of the present invention; as can be seen from fig. 5, the intensity of luminescence is weakened, indicating that energy transfer between donor and receptor occurs, resulting from specific binding of antigen/antibody.
FIG. 6 is a graph showing the change of luminescence intensity of the up-conversion nanomaterial after performing an immune competition reaction by introducing a solution containing an antigen (goat IgG, 40. mu.g/mL) in example 1 of the present invention. From fig. 6, it can be seen that the luminescence becomes stronger due to the reduction of the number of donor-acceptor pairs where energy transfer occurs and the luminescence of the rare earth luminescent nanocrystal is restored.
Example 2
Steps 1), 3), 4), 5) are the same as in example 1, with 2) of example 1 being replaced by:
1mmol of rare earth chloride (including 0.745mmol YCl)3·6H2O，0.25mmolYbCl3·6H2O and 0.05mmol TmCl3·6H2O) was placed in a three-necked flask having a capacity of 50mL, and 15mL of octadecene and 6mL of oleic acid were added. And then putting the three-necked bottle into a magnetic stirring heater, introducing argon for protection, heating to 160 ℃, and obtaining a yellowish, clear and transparent solution after the chloride is completely dissolved. Then cooled to room temperature, 4mmol of NH will dissolve4F. Dropwise adding a 10mL methanol solution of 2.5mmol of LiOH into the three-necked bottle, heating to 75 ℃, keeping the temperature for 30min, completely removing the methanol in the solution, heating to 300 ℃, reacting for 1.5h, keeping introducing argon for protection in the reaction process, refluxing by using cooling water, cooling to room temperature after the reaction is finished, adding ethanol, centrifuging and purifying for three times to obtain RENCs, and dissolving the obtained RENCs into 8mL cyclohexane for later use.
The results of using scanning electron microscopy for detecting RENCs are shown in FIG. 7. FIG. 7 is a scanning electron micrograph of RENCs prepared in example 2, and it can be seen from FIG. 7 that RENCs obtained were uniform particles having a particle size of about 25 nm.
The emission spectrum of the obtained antibody-labeled RENCs with 980nm excitation light was detected, and the emission spectrum in the wavelength range of 300 to 850nm was measured as shown in FIG. 8. As can be seen from FIG. 8, RENCs emit light in the 800nm near infrared region together with Bi2Se3There is a large overlap of the absorption spectra (fig. 2) indicating an efficient energy transfer detection application.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. A test composition characterized by having a first and a second concentration of a compound,
comprises a bismuth-based material marked with an antigen and a rare earth doped luminescent nanocrystal marked with an antibody;
alternatively, the first and second electrodes may be,
comprises a bismuth-based material marked with an antibody and a rare earth doped luminescent nanocrystal marked with an antigen;
the antigen and the antibody are an antigen-antibody pair which can generate specific binding reaction;
the bismuth-based material is a bismuth-based two-dimensional nano sheet material or a metal ion-doped bismuth-based two-dimensional nano sheet material.
2. The assay composition of claim 1,
the molar ratio of the bismuth-based material marked with the antigen to the rare earth doped luminescent nanocrystal marked with the antibody is 1 (1-10);
the molar ratio of the bismuth-based material marked with the antibody to the rare earth-doped luminescent nanocrystal marked with the antigen is 1 (1-10).
3. The assay composition of claim 2,
the molar ratio of the bismuth-based material marked with the antigen to the rare earth doped luminescent nanocrystal marked with the antibody is 1: 1;
the molar ratio of the bismuth-based material marked with the antibody to the rare earth doped luminescent nanocrystal marked with the antigen is 1: 1.
4. The detection composition as claimed in claim 1, wherein the bismuth-based two-dimensional nano-sheet material is Bi2S3、Bi2Se3Or Bi2Te3。
5. The assay composition of claim 1,
the preparation method of the bismuth-based material marked with the antigen comprises the following steps:
mixing the bismuth-based material with a solution containing the antigen overnight, centrifuging, and dispersing the obtained precipitate in a buffer solution to obtain the solution of the bismuth-based material marked with the antigen.
The preparation method of the bismuth-based material marked with the antibody comprises the following steps:
mixing the bismuth-based material with the solution containing the antibody overnight, centrifuging, and dispersing the obtained precipitate in a buffer solution to obtain the solution of the bismuth-based material marked with the antibody.
6. The assay composition of claim 1,
the preparation method of the rare earth doped luminescent nanocrystal marked with the antibody comprises the following steps:
coating a carboxyl polymer on the rare earth doped luminescent nanocrystal, then mixing the coated rare earth doped luminescent nanocrystal with an activator and a solution containing an antibody, reacting, centrifuging, and dispersing the obtained precipitate in a buffer solution to obtain a solution of the rare earth doped luminescent nanocrystal marked with the antibody;
the preparation method of the rare earth doped luminescent nanocrystal marked with the antigen comprises the following steps:
and (2) coating the rare earth doped luminescent nanocrystal with a carboxyl polymer, then mixing the coated rare earth doped luminescent nanocrystal with an activator and a solution containing an antigen, reacting, centrifuging, and dispersing the obtained precipitate in a buffer solution to obtain the solution of the rare earth doped luminescent nanocrystal marked with the antigen.
7. A method for detecting an antigen or an antibody using the detection composition according to any one of claims 1 to 6, comprising the steps of:
1) uniformly mixing the bismuth-based material solution marked with the antigen and the rare earth-doped luminescent nanocrystal solution marked with the antibody to obtain a detection system;
2) and adding a sample to be detected into the detection system, and performing emission spectrum detection.
Alternatively, the first and second electrodes may be,
1) uniformly mixing the bismuth-based material solution marked with the antibody and the rare earth-doped luminescent nanocrystal solution marked with the antigen to obtain a detection system;
2) and adding a sample to be detected into the detection system, and performing emission spectrum detection.
8. The detection method as claimed in claim 7, wherein the excitation light of the emission spectrum is near infrared light of 700-1700 nm, and the emission light is visible light of 400-800 nm or near infrared light of 900-1700 nm.
9. The method of detecting according to claim 8,
the excitation light of the emission spectrum is near-infrared light of 800nm, and the emission light is visible light of 540nm, visible light of 650nm, near-infrared light of 1064nm or near-infrared light of 1530 nm;
alternatively, the first and second electrodes may be,
the excitation light of the emission spectrum is near-infrared light of 980nm, and the emission light is 690nm visible light, 800nm visible light, 1064nm near-infrared light, 1180nm near-infrared light or 1530nm near-infrared light;
alternatively, the first and second electrodes may be,
the excitation light of the emission spectrum is 1530nm near infrared light, and the emission light is 650nm visible light, 800nm visible light or 1180nm near infrared light.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111443663.7A CN114137217A (en) | 2021-11-30 | 2021-11-30 | FRET-based homogeneous immunoassay method and detection composition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111443663.7A CN114137217A (en) | 2021-11-30 | 2021-11-30 | FRET-based homogeneous immunoassay method and detection composition |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114137217A true CN114137217A (en) | 2022-03-04 |
Family
ID=80390005
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111443663.7A Pending CN114137217A (en) | 2021-11-30 | 2021-11-30 | FRET-based homogeneous immunoassay method and detection composition |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114137217A (en) |
-
2021
- 2021-11-30 CN CN202111443663.7A patent/CN114137217A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104804741B (en) | Single-emission up-conversion nano fluorescent probe and synthetic method thereof | |
| Zhou et al. | Efficient dual-modal NIR-to-NIR emission of rare earth ions co-doped nanocrystals for biological fluorescence imaging | |
| Wang et al. | Microwave-assisted synthesis of carbon nanodots through an eggshell membrane and their fluorescent application | |
| Huang et al. | Rare earth ion–and transition metal ion–doped inorganic luminescent nanocrystals: from fundamentals to biodetection | |
| Bouzigues et al. | Biological applications of rare-earth based nanoparticles | |
| CN111175266B (en) | Construction method and detection method of near-infrared fluorescence biosensor | |
| Bhuckory et al. | Core or shell? Er3+ FRET donors in upconversion nanoparticles | |
| WO2003074630A1 (en) | Fine particle containing rare earth element and fluorescent probe using the same | |
| CN106867509A (en) | A kind of Nd3+Conversion nano crystalline substance material and preparation method thereof and water detect application on sensitization nucleocapsid | |
| CN107033905A (en) | A kind of rear-earth-doped lithium yttrium fluoride nano material and preparation method and application | |
| CN109468128B (en) | Graphene quantum dot-rare earth up-conversion nano composite material and preparation method and application thereof | |
| CN105505392B (en) | Rare earth oxyfluoride nano material and its preparation method and application | |
| CN111426658B (en) | Method for detecting thrombin by utilizing quantum dot sensitized up-conversion nano material | |
| CN107356570B (en) | Solid-state up-conversion fluorescent probe and preparation method and application thereof | |
| CN113817469B (en) | Ultra-bright monochromatic up-conversion nano probe for excitation/emission in biological window and preparation method and application thereof | |
| KR101701885B1 (en) | A composite compound comprising upconverting nanoparticle and specific receptor | |
| CN111234556A (en) | Near-infrared cyanine dye-sensitized up-conversion luminescence nano probe and construction method thereof | |
| Ning et al. | Manganese-mediated growth of ZnS shell on KMnF3: Yb, Er cores toward enhanced up/downconversion luminescence | |
| CN102517019A (en) | Method for improving photoluminescence efficiency of upconversion material | |
| CN111234819B (en) | Rare earth doped nano probe and preparation thereof and new coronavirus detection probe | |
| CN106701066B (en) | A kind of preparation method and applications of blue emission carbon quantum dot | |
| KR101423589B1 (en) | Non-toxic Fluorescent particle including lanthanide complexes | |
| CN114675026A (en) | Dissolution-enhanced long afterglow luminescence detection method | |
| CN105754600B (en) | A kind of core-shell structure copolymer-shell structural nano fluorescence probe of europium ion activation and its preparation method and application | |
| A Shivkumar et al. | FRET from CdSe/ZnS core-shell quantum dots to fluorescein 27 dye |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |