CN111735964A - Single-molecule immunodetection method based on up-conversion fluorescent probe - Google Patents

Single-molecule immunodetection method based on up-conversion fluorescent probe Download PDF

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CN111735964A
CN111735964A CN202010587667.1A CN202010587667A CN111735964A CN 111735964 A CN111735964 A CN 111735964A CN 202010587667 A CN202010587667 A CN 202010587667A CN 111735964 A CN111735964 A CN 111735964A
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何皓
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Shenzhen Guanghe Biotechnology Co Ltd
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Abstract

The invention belongs to the technical field of immunodetection, and particularly relates to a monomolecular immunodetection method based on an up-conversion fluorescent probe, wherein a sample to be detected is diluted and then is dripped onto a detection substrate, a detected object is combined with a bioactive molecule B, the detection substrate is washed after the completion, then the diluted immunofluorescence probe is dripped to ensure that the bioactive molecule A is combined with the detected object combined with the bioactive molecule B, and the detection substrate is washed after the completion; and (3) placing the processed detection substrate under a fluorescence microscope, and counting the number of the immunofluorescence probes, wherein the obtained number is the number of the detected objects. Compared with the prior art, the invention has the following advantages: the high-concentration rare earth-doped up-conversion luminescent nano-particles are used as the basis of the immunofluorescence probe, the result is readable by naked eyes under a fluorescence microscope, and the quantity of the immunofluorescence probe can be determined, so that the detection method is suitable for pathogens, microorganisms, macromolecular antigens or antibodies, and the detection range is wide.

Description

Single-molecule immunodetection method based on up-conversion fluorescent probe
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a monomolecular immunodetection method based on an up-conversion fluorescent probe.
Background
The immunoassay plays an important role in the field of detection of various diseases, the existing chemiluminescence technology cannot realize single-molecule immunoassay, and the bottleneck of the lower limit of the detection sensitivity comes from the uncertainty of a molecular level; acridine chemiluminescence systems are widely applied to the fields of inorganic organic compounds, biology, drug analysis and the like because of the advantages of no need of catalysts, mild reaction conditions, good reproducibility and the like, acridine esters can release a plurality of photons within a very short time when encountering a luminescence excitation substrate, and the subsequent structure is rapidly destroyed to lose the luminous capacity; because the photon direction of the acridinium ester is uncontrollable when the acridinium ester releases photons, and a photomultiplier tube (PMT) serving as a core component of the luminescence detection device is directional, a few photons released by a single acridinium ester cannot be detected by the PMT, and meanwhile, a photon signal captured on the PMT cannot trace the source of a single acridinium ester molecule in a solution; when the concentration of antigen molecules in a sample is low to a certain degree, the uncertainty of the quantity of photons captured by the PMT is exponentially increased, so that a detection signal is completely buried in background noise, the quantitative detection capability is lost, and the same problem also exists in an electrochemical luminescence system and an enzymatic chemiluminescence system. Therefore, today that the automation technology of the apparatus has been developed to a considerable level, the detection sensitivity of the existing chemiluminescence technology has been close to its theoretical detection limit level, and cannot reach the single molecule detection level. The immunoassay plays an important role in various disease monitoring fields, along with the progress of the technology, the requirement on the sensitivity of immunoassay is higher and higher, the sensitivity of the traditional mainstream chemiluminescence technology is difficult to realize single-molecule level detection, and the detection of part of target molecules with extremely low concentration is directly caused to be an incompletable task, so how to improve the fluorescence signal intensity of the probe on the basis of the prior art so as to realize the technical effect that a single probe can be easily detected.
The upconversion nanometer material is an inorganic nanometer material doped with rare earth ions, can continuously wash two or more near-far infrared photons (generally 980 nm) of hands and emit high-energy ultraviolet-visible light, can effectively avoid interference of autofluorescence and scattered light of a biological sample due to the near-infrared excitation property of the upconversion nanometer material, can effectively reduce light damage of ultraviolet or visible light excitation on the biological sample, can improve sensitivity and signal-to-noise ratio in immunoassay, but is poor in water solubility and dispersibility, so that the upconversion rare earth nanometer material needs to be coupled with biomolecules after chemical modification, and the problem of low sensitivity of the existing mainstream chemiluminescence technology is solved.
Disclosure of Invention
The invention aims to provide a monomolecular immunoassay method based on an up-conversion fluorescent probe, aiming at the problem that the sensitivity of the existing mainstream chemiluminescence technology cannot realize monomolecular level detection.
The invention is realized by the following technical scheme: a monomolecular immunoassay method based on an up-conversion fluorescent probe comprises the immunofluorescent probe, a detection substrate and a sample to be detected;
the immunofluorescence probe is obtained by modifying the surface of nano material particles with high-concentration rare earth doping to modify a bioactive molecule A required by immunodetection;
the detection substrate is used for capturing a biological active molecule B of a detected object by modifying the substrate;
the detected sample contains a detected object which is a pathogen, a microorganism, a macromolecular antigen or an antibody;
when the detected object is a pathogen, a microorganism or a macromolecular antigen, the bioactive molecule A is an antibody A capable of being specifically combined with the detected object, and the bioactive molecule B is an antibody B capable of being specifically combined with the detected object; when the detected object is an antibody, the bioactive molecule A is the antibody A which can be specifically recognized by the detected object, and the bioactive molecule B is an antigen which can be specifically recognized by the detected object;
during detection, diluting a sample to be detected by using a slow release solution, dripping the sample to be detected on a detection substrate, combining a detected object with a bioactive molecule B, flushing the detection substrate after the detection is finished, dripping an immunofluorescence probe diluted by using the slow release solution to combine the bioactive molecule A with the detected object combined with the bioactive molecule B, flushing the detection substrate after the detection is finished, and washing away the unbound immunofluorescence probe; and (3) placing the processed detection substrate under a fluorescence microscope, using near infrared light as an excitation light source and visible light wave bands as detection wave bands, carrying out fluorescence imaging on the whole detection substrate, and counting the number of the immunofluorescence probes, wherein the obtained number is the number of the detected objects in the detected sample.
Specifically, the rare earth doping proportion in the high-concentration rare earth doped up-conversion nano material particles is more than 2%, the high-concentration rare earth doping enables the up-conversion nano material particles to have larger anti-stokes displacement, compared with the traditional organic dye, an excitation light source and an emission waveband are not overlapped, the spontaneous fluorescence background noise can be effectively inhibited, and the signal to noise ratio of a detection signal can be obviously improved; the particle size of the high-concentration rare earth doped up-conversion nano material particles is 10-100 nm; the detection substrate is modified after the aldehyde modification of the substrate, so that the effective combination of the biological active molecules B can be ensured; the substrate is a glass sheet or a silicon wafer;
the wavelength of the laser light source is 790nm, 980nm or 1550nm, and the energy density of the laser light source passing through a microscope light path is larger than 1W/cm.
Specifically, the immunofluorescence probe is NaYF4、NaGdF4、CaF2、LiYF4、NaLuF4、LiLuF4、KMnF3Or Y2O3The fluorescent probe is a luminescent substrate, is co-doped with a sensitizer and an activator to obtain up-conversion fluorescent nanoparticles UCNPs, then the surface of the UCNPs particles is subjected to carboxylation modification to obtain carboxylation modified nanoparticles UCNP @ PEG-COOH, and then the fluorescent probe is obtained through modification of bioactive molecules A;
the slow release solution is a PB buffer solution with the mass concentration of 0.2mol/L, and solutes in the PB buffer solution comprise 0.1% BSA, 2% sucrose and 0.1% Tween according to mass percentage.
Compared with the prior art, the invention has the following advantages: the high-concentration rare earth-doped up-conversion luminescence nanoparticles are used as the basis of the immunofluorescence probe, the luminescence intensity is greatly improved, the result can be read by naked eyes under a fluorescence microscope, the quantity can be quantified through the immunofluorescence probe, the method is suitable for pathogens, microorganisms, macromolecular antigens or antibodies respectively according to different immunization modes, and the detectable range is wide.
Drawings
FIG. 1 is a schematic diagram of the structure of an immunofluorescent probe.
FIG. 2 is a structural view of a sandwich mode in example 1.
FIG. 3 is a structural view of a sandwich mode in example 2.
FIG. 4 is an image of the fluorescent probe in the microscope at the first detection time for the 0.01 pg/mL concentration of the experimental standard.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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 invention.
The most key concept of the invention is as follows: the high-concentration rare earth doped up-conversion luminescence nano-particles are used as the basis of the immunofluorescence probe, and the result can be read under a fluorescence microscope and can be quantified through the number of the fluorescence probes; according to different immune reaction modes, the method can be divided into a sandwich mode and an indirect mode; the sandwich mode-based single-molecule immunoassay method can be used for detecting pathogens, microorganisms, macromolecular antigens, antibodies and the like existing in a sample, wherein the mode for detecting the pathogens, the microorganisms and the macromolecular antigens is a double-antibody sandwich method, and the mode for detecting the antibodies is a double-antigen sandwich method; single molecule immunoassays based on the indirect mode can be used to detect antibodies in a sample; and carrying out fluorescence imaging on the whole detection substrate, and counting the number of the immunofluorescence probes, wherein the obtained number is the number of the detected objects in the detected sample.
Example 1
As shown in fig. 1-2, a single-molecule immunoassay method based on an up-conversion fluorescent probe, which is a sandwich mode in this embodiment, includes an immunofluorescent probe, a detection substrate and a sample to be detected;
the immunofluorescence probe is obtained by modifying the surface of the high-concentration rare earth doped up-conversion nano material particles 1 with bioactive molecules A required by immunodetection, wherein the bioactive molecules A are antibodies A2 capable of being specifically combined with an object to be detected;
the detection substrate is used for capturing a biological active molecule B of a detected object by modifying the substrate 3, and the biological active molecule B is an antibody B4 capable of being specifically combined with the detected object;
the detected sample contains a detected object, and the detected object is a macromolecular antigen 5;
wherein,
the detection of prostate specific antigen PSA in this example includes:
first, reagent preparation
Modifying a bioactive molecule A (PSA monoclonal antibody) on the surface of a high-concentration rare earth doped up-conversion nano material particle, and modifying an antibody B4 (PSA polyclonal antibody) capable of being specifically combined with a detected object on the surface of a substrate 3;
second, quantitative detection
Diluting a detected sample, namely a PSA standard substance, to the following concentration by using a PB buffer solution with the mass concentration of 0.2mol/L to obtain an experimental standard substance: 0.01 pg/mL, 0.1 pg/mL, 1 pg/mL, 10 pg/mL;
0.5 microliter of each group of experimental standard is respectively detected for three times, the counting of the fluorescent probe in the microscope imaging in the three times of detection is averaged, the imaging of the fluorescent probe in the microscope is carried out on the experimental standard with the concentration of 0.01 pg/mL in the figure 4 in the first detection, the bright point in the original image is blue, the bright point is a modified figure and is only shown schematically and not taken as a statistical standard.
The results of counting the average values after multiple measurements of each concentration are shown in table 1:
TABLE 1
Concentration of Number of standard molecules Fluorescent probe readings
0.01 pg/mL ≈ 103 1025
0.1 pg/mL ≈ 104 10586
1 pg/mL ≈ 105 109815
10 pg/mL ≈ 106 1140288
The number of prostate specific antigen PSA was obtained as shown in table 1.
Example 2
As shown in fig. 3, based on example 1, a single-molecule immunoassay method based on an up-conversion fluorescent probe, which is a sandwich mode in this embodiment, includes an immunofluorescent probe, a detection substrate and a sample to be detected;
the immunofluorescence probe is obtained by modifying the surface of the high-concentration rare earth doped up-conversion nano material particles 1 with bioactive molecules A required by immunodetection, wherein the bioactive molecules A are antigens A2 capable of being specifically combined with an object to be detected;
the detection substrate is used for capturing a biological active molecule B of a detected object by modifying the substrate 3, and the biological active molecule B is an antigen B6 capable of being specifically combined with the detected object;
the sample to be detected contains a detected object, and the detected object is an antibody 7;
the preparation method of the nano fluorescent probe comprises the following steps:
(1) upconversion fluorescent nanoparticle preparation
Mixing 1mmol of RECl3 solution, 6mL of oleic acid and 15mL of octadecene, adding the mixture into a 100mL three-neck round-bottom flask, stirring and heating to 160 ℃ under the protection of argon flow, and maintaining for 20min to obtain a clear primary mixed solution; RE in RECl3 solution3+Comprising Y3+、Nd3+And Ce3+Eu, an activator3+Wherein Nd is3+And Ce3+In a molar ratio of 3: 1; the RECl3 solution RE3+The mol percent of the medium sensitizer is 50mol percent, and the mol percent of the activator is 6mol percent;
cooling the primary mixed solution to 50 ℃, adding 10mL of methanol solution containing 4mmol of ammonium fluoride and 2.5mmol of sodium hydroxide, then heating to 150 ℃, and maintaining for 20min to remove methanol to obtain a secondary mixed solution;
heating the secondary mixed solution to 310 ℃ and maintaining for 90min, thermally injecting an oleic acid/octadecene solution containing sodium trifluoroacetate and yttrium trifluoroacetate into a reaction system, wherein the volume ratio of oleic acid to octadecene is 1:1, maintaining for 60min at 310 ℃, cooling the liquid to room temperature after the reaction is finished, centrifugally washing with ethanol and cyclohexane, and drying at 58 ℃ to obtain UCNPs;
(2) preparation of carboxyl modified nano-particles
Adding 1mL of UCNPs cyclohexane solution with the concentration of 10mM into 1mL of hydrochloric acid solution with the concentration of 0.3mol/L, carrying out ultrasonic treatment for 10min, stirring for 3h to obtain a mixed solution, then centrifuging the mixed solution at a high speed, removing a supernatant to obtain a first precipitate, washing the first precipitate for 5 times to obtain acid-washed UCNPs, and dispersing the acid-washed UCNPs into 1mL of deionized water to obtain an acid-washed UCNPs dispersion liquid;
mixing the obtained acid-washed UCNPs dispersion with a proper amount of PEG-COOH, stirring for 24 hours at room temperature, centrifuging to obtain a second precipitate, washing the second precipitate with water for 5 times to remove redundant PEG-COOH, and obtaining UCNP @ PEG-COOH;
(3) fluorescent probe preparation
Dispersing UCNP @ PEG-COOH into 1mL of deionized water to obtain a UCNP @ PEG-COOH dispersion liquid, adding the UCNP @ PEG-COOH dispersion liquid into a PB buffer solution with the pH value of 7.2 and the concentration of 0.2mol/L to prepare suspension liquid with the concentration of 2mg/mL, then, NHS with the concentration of 50mg/mL and EDC with the concentration of 50mg/mL are added into the suspension in sequence, so that the mass ratio of the NHS to the luminescent substrate is 1:3, the mass ratio of the EDC to the UCNPs is 1:5, then reacting for 30min at the temperature of 8 ℃, centrifuging to remove redundant NHS and EDC, adding 0.2mol/L PB buffer solution to obtain a suspension II, adding bioactive molecule A into the suspension II, reacting for 2 hours at the temperature of 22 ℃, wherein the mass ratio of the bioactive molecule A to the UCNPs is 1:300, and adding BSA solution with the mass concentration of 2% for sealing for 1 hour after the reaction is finished; after that, centrifugally washing for 2 times at the temperature of 3 ℃ to obtain a UCNPs-bioactive molecule A conjugate which is a fluorescent probe, storing the UCNPs-bioactive molecule A conjugate in PB buffer solution with the concentration of 0.02mol/L, and storing for later use at the temperature of 5 ℃;
the detection substrate is a modified bioactive molecule B obtained by amination treatment of a glass sheet.
During detection, diluting a sample to be detected by using a slow release solution, dripping the sample to be detected on a detection substrate, combining a detected object with a bioactive molecule B, flushing the detection substrate after the detection is finished, dripping an immunofluorescence probe diluted by using the slow release solution to combine the bioactive molecule A with the detected object combined with the bioactive molecule B, flushing the detection substrate after the detection is finished, and washing away the unbound immunofluorescence probe; and (3) placing the processed detection substrate under a fluorescence microscope, using near infrared light as an excitation light source and visible light wave bands as detection wave bands, carrying out fluorescence imaging on the whole detection substrate, and counting the number of the immunofluorescence probes, wherein the obtained number is the number of the detected objects in the detected sample.
Example 3
On the basis of embodiment 1, a monomolecular immunoassay method based on an up-conversion fluorescent probe, which is an indirect mode in this embodiment, comprises the immunofluorescent probe, a detection substrate and a sample to be detected;
the immunofluorescence probe is obtained by modifying the surface of the nano material particles 1 with high-concentration rare earth doping with bioactive molecules A required by immunodetection, wherein the bioactive molecules A are antibodies A which can be specifically identified by a detected object;
the detection substrate is used for capturing a biological active molecule B of an object to be detected by modifying the substrate 3, and the biological active molecule B is an antigen which can be specifically recognized by the object to be detected;
the sample to be detected contains a detected object, and the detected object is an antibody.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (8)

1. A monomolecular immunoassay method based on an up-conversion fluorescent probe is characterized by comprising the immunofluorescence probe, a detection substrate and a sample to be detected;
the immunofluorescence probe is obtained by modifying the surface of nano material particles with high-concentration rare earth doping to modify a bioactive molecule A required by immunodetection;
the detection substrate is used for capturing a biological active molecule B of a detected object by modifying the substrate;
the detected sample contains a detected object which is a pathogen, a microorganism, a macromolecular antigen or an antibody;
when the detected object is a pathogen, a microorganism or a macromolecular antigen, the bioactive molecule A is an antibody A capable of being specifically combined with the detected object, and the bioactive molecule B is an antibody B capable of being specifically combined with the detected object; when the detected object is an antibody, the bioactive molecule A is the antibody A which can be specifically recognized by the detected object, and the bioactive molecule B is an antigen which can be specifically recognized by the detected object;
during detection, diluting a sample to be detected by using a slow release solution, dripping the sample to be detected on a detection substrate, combining a detected object with a bioactive molecule B, flushing the detection substrate after the detection is finished, dripping an immunofluorescence probe diluted by using the slow release solution to combine the bioactive molecule A with the detected object combined with the bioactive molecule B, flushing the detection substrate after the detection is finished, and washing away the unbound immunofluorescence probe; and (3) placing the processed detection substrate under a fluorescence microscope, using near infrared light as an excitation light source and visible light wave bands as detection wave bands, carrying out fluorescence imaging on the whole detection substrate, and counting the number of the immunofluorescence probes, wherein the obtained number is the number of the detected objects in the detected sample.
2. The single-molecule immunoassay method based on the upconversion fluorescent probe according to claim 1, wherein the rare earth doping ratio in the high-concentration rare earth-doped upconversion nanomaterial particles is greater than 2%.
3. The single-molecule immunoassay method based on the upconversion fluorescent probe according to claim 1, wherein the particle size of the high-concentration rare earth-doped upconversion nanomaterial particles is 10-100 nm.
4. The method for the single-molecule immunoassay based on the up-conversion fluorescent probe of claim 1, wherein the detection substrate is modified after the substrate is aldehyde modified.
5. The method for single-molecule immunoassay based on the upconversion fluorescent probe according to claim 4, wherein the substrate is a glass sheet or a silicon wafer.
6. The method for single-molecule immunodetection based on an upconversion fluorescent probe according to claim 1, wherein the wavelength of the laser light source is 790nm, 980nm or 1550nm, and the energy density of the laser light source passing through a microscope optical path is greater than 1W/cm.
7. The method for single-molecule immunoassay based on up-conversion fluorescent probe of claim 1, wherein the immunofluorescent probe is NaYF4、NaGdF4、CaF2、LiYF4、NaLuF4、LiLuF4、KMnF3Or Y2O3The fluorescent probe is a luminescent substrate, is co-doped with a sensitizer and an activator to obtain up-conversion fluorescent nanoparticles UCNPs, then is subjected to carboxylation modification on the surface of the UCNPs particles to obtain carboxylation modified nanoparticles UCNP @ PEG-COOH, and is modified by bioactive molecules A to obtain the fluorescent probe.
8. The single-molecule immunoassay method based on the up-conversion fluorescent probe of claim 1, wherein the slow-release solution is a PB buffer solution with a mass concentration of 0.2mol/L, and solutes in the PB buffer solution comprise 0.1% BSA, 2% sucrose and 0.1% Tween according to mass percentage.
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