阅读说明：本技术 一种基于上转换荧光探针的核酸快速检测方法 (Rapid nucleic acid detection method based on up-conversion fluorescent probe ) 是由 何皓 于 2020-06-24 设计创作，主要内容包括：本发明属于核酸检测技术领域,具体涉及一种基于上转换荧光探针的核酸快速检测方法,用于检测目标样本中核酸分子的数目,将目标样本处理得到DNA单链样品,稀释后滴加到检测基板上进行捕获杂交,完成后,冲洗检测基板,将荧光探针稀释后滴加到前述检测基板,完成后冲洗检测基板；将冲洗后的检测基板置于荧光显微镜下,对荧光探针数目进行计数,所得数目即为目标样本中核酸分子的数目。本发明相比现有技术具有以下优点：根据目标核酸片段设计对应的捕获核酸片段和标记核酸片段,能够实现对含有不同目标核酸片段的目标样本中核酸分子数量进行检测,适用范围广、准确度高,可以通过肉眼观测到核酸分子并进行定量。(The invention belongs to the technical field of nucleic acid detection, and particularly relates to a rapid nucleic acid detection method based on an up-conversion fluorescent probe, which is used for detecting the number of nucleic acid molecules in a target sample, processing the target sample to obtain a DNA single-stranded sample, diluting the DNA single-stranded sample, dropwise adding the diluted DNA single-stranded sample onto a detection substrate for capture hybridization, flushing the detection substrate after the completion, diluting the fluorescent probe, dropwise adding the diluted fluorescent probe onto the detection substrate, and flushing the detection substrate after the completion; and (3) placing the washed detection substrate under a fluorescence microscope, and counting the number of the fluorescence probes, wherein the obtained number is the number of the nucleic acid molecules in the target sample. Compared with the prior art, the invention has the following advantages: the corresponding capture nucleic acid fragment and the corresponding marker nucleic acid fragment are designed according to the target nucleic acid fragment, so that the number of nucleic acid molecules in a target sample containing different target nucleic acid fragments can be detected, the application range is wide, the accuracy is high, and the nucleic acid molecules can be observed by naked eyes and quantified.)

1. A nucleic acid rapid detection method based on an up-conversion fluorescent probe is used for detecting the number of nucleic acid molecules in a target sample and is characterized by comprising the fluorescent probe obtained by modifying up-conversion fluorescent nanoparticles by a labeled nucleic acid fragment, a detection substrate modified by a capture nucleic acid fragment and the target sample; the target sample comprises target nucleic acid fragments, the capture nucleic acid fragments can be matched and hybridized with a part of the target nucleic acid fragments, and the marker nucleic acid fragments can be matched and hybridized with a part of the target nucleic acid fragments which cannot be matched and hybridized with the capture nucleic acid fragments;

the detection method comprises the following steps:

(1) diluting a target sample by using a PB buffer solution to obtain an experimental standard substance with corresponding concentration, uniformly dripping the experimental standard substance onto a detection substrate, washing the detection substrate after the target sample is combined with a captured nucleic acid fragment on the detection substrate, and washing away the unbound target sample;

the preparation method of the target sample comprises the following steps: carrying out nucleic acid extraction on a target sample to be detected to obtain a target nucleic acid fragment, and dissociating the target nucleic acid fragment into a DNA single-stranded sample, wherein the DNA single-stranded sample is the target sample;

(2) diluting the fluorescent probe with a PB buffer solution to obtain a fluorescent probe diluent, uniformly dropwise adding the fluorescent probe diluent to the surface of the detection substrate treated in the step (1) for hybridization, washing the detection substrate, and washing away the unbound fluorescent probe;

(3) and (3) placing the detection substrate processed in the step (2) 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 fluorescent probes, wherein the obtained number is the number of the nucleic acid molecules in the target sample.

2. The method for rapidly detecting nucleic acid based on up-conversion fluorescent probe as claimed in claim 1, wherein the method comprisesCharacterized in that the fluorescent 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 the labeled nucleic acid fragments are subjected to amination treatment and then are modified to obtain the fluorescent probe.

3. The method for rapidly detecting nucleic acid based on the up-conversion fluorescent probe as claimed in claim 2, wherein the preparation method of the fluorescent probe comprises:

(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⁺A sensitizer, an activator;

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 52-58 ℃ to obtain UCNPs;

(2) preparation of carboxyl modified nano-particles

Adding 1mL of 10mM UCNPs cyclohexane solution into 1mL of 0.3mol/L hydrochloric acid solution, performing ultrasonic treatment for 10min, stirring for 3h to obtain a mixed solution, centrifuging the mixed solution at a high speed, removing a supernatant to obtain a first precipitate, washing the first precipitate for 5-8 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 3-5 times to remove the redundant PEG-COOH, and obtaining UCNP @ PEG-COOH;

(3) fluorescent probe preparation

Dispersing UCNP @ PEG-COOH into 1mL of deionized water to obtain UCNP @ PEG-COOH dispersion liquid, adding the UCNP @ PEG-COOH dispersion liquid into 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 1-2mg/mL, then adding NHS with the concentration of 50mg/mL and EDC with the concentration of 50mg/mL into the suspension liquid in sequence to ensure that the mass ratio of the NHS to the luminescent substrate is 1:2-5 and the mass ratio of the EDC to the UCNPs is 1:2-5, then reacting for 20-60min at the temperature of 4-30 ℃, centrifuging to remove redundant NHS and EDC, then adding 0.2mol/L of PB buffer solution to obtain a second suspension liquid, adding an amination labeled nucleic acid fragment into the second suspension liquid, reacting for 1-3 hours at the temperature of 4-30 ℃, the mass ratio of the labeled nucleic acid fragment to the UCNPs is 1:100-1000, and BSA solution with the mass concentration of 0.5-2% is added for blocking for 1-2 hours after the reaction is finished; and after the reaction is finished, centrifugally washing for 1-2 times at the temperature of 3-5 ℃ to obtain a UCNPs-labeled nucleic acid fragment conjugate, namely the fluorescent probe, storing the UCNPs-labeled nucleic acid fragment conjugate in a PB buffer solution with the concentration of 0.02mol/L, and storing at the temperature of 2-8 ℃ for later use.

4. The method for rapidly detecting nucleic acid based on up-conversion fluorescent probe as claimed in claim 3, wherein RECl3 solution RE³⁺The mol percentage of the mesosensitizer is 15mol%, 20mol%, 25mol%, 30mol%, 35mol%, 40mol%, 45mol%, 50mol%, 55mol%, 60mol%, 65mol%, 70mol% or 75 mol%; RECl3 solution RE³⁺The molar percentage of the medium activator is 2.5mol%, 3mol%, 4mol%, 5mol%, 6mol%, 7mol% or 8 mol%.

5. The method for rapidly detecting nucleic acid based on the upconversion fluorescent probe as claimed in claim 3, wherein the particle size of the upconversion nanoparticle is 25-200 nm.

6. The method for rapidly detecting nucleic acid based on the up-conversion fluorescent probe as claimed in claim 3, wherein the solutes in the PB buffer comprise 0.1% BSA, 2% sucrose, and 0.1% Tween by mass.

7. The method for rapidly detecting nucleic acid based on up-conversion fluorescent probe of claim 2, wherein the sensitizer is Yb³⁺、Nd³⁺、Gd³⁺、Ce³⁺One or more of them are mixed; the activator is Yb³⁺、Er³⁺、Tm³⁺、Sm³⁺、Dy³⁺、Ho³⁺、Eu³⁺、Tb³⁺、Pr³⁺One or more of them are mixed.

8. The method for rapidly detecting nucleic acid based on the upconversion fluorescent probe according to claim 1, wherein when the target nucleic acid fragment is RNA, the target nucleic acid fragment is subjected to reverse transcription to obtain a DNA double strand, and then the DNA double strand is dissociated into a DNA single strand sample; when the target nucleic acid fragment is a DNA double strand, the target nucleic acid fragment is dissociated into a DNA single strand sample.

9. The method for rapidly detecting nucleic acid based on the up-conversion fluorescent probe as claimed in claim 1, wherein the substrate is a glass sheet or a silicon wafer.

10. The method for rapidly detecting nucleic acid based on the 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 the microscope optical path is greater than 1W/cm.

Technical Field

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a rapid nucleic acid detection method based on an up-conversion fluorescent probe.

Background

Nucleic acid mainly comprises two major categories of deoxyribonucleic acid and ribonucleic acid, is important biomacromolecule, is a carrier of genetic information, is important content of molecular biology and life science research, and the composition, the arrangement sequence, the structural characteristics and the biological function of the nucleic acid are the most important research content of the nucleic acid, but for the quantitative determination of the nucleic acid, the quantitative determination is still one of the subjects concerned and researched by researchers, and the determination has immeasurable significance for researching the biochemical reaction of the nucleic acid, developing nucleic acid medical products and diagnosing and preventing diseases; there are many methods for quantitatively determining nucleic acids, such as chromatography, spectrophotometry, fluorometry, which is a common method for quantitatively determining nucleic acids, but indirect determination with a fluorescent probe is required because of low fluorescence quantum yield of nucleic acids, microscopy, immunoassay, and resonance rayleigh scattering; when the suspected infected person is faced, diagnosis can be quickly and accurately made, the infected person can be effectively isolated, and the important effect on epidemic situation prevention and control is achieved. In the epidemic prevention battle, the nucleic acid detection becomes the gold standard for the confirmed diagnosis of cases, and becomes the strongest powerful weapon in the epidemic prevention work; however, no matter the traditional fluorescent quantitative PCR technology or the high-end digital PCR or constant-temperature PCR technology, the nucleic acid detection technology needs to amplify a target nucleic acid fragment for detection, because nucleic acid molecules are small and can only be combined with limited signal probes, the existing fluorescent probes mainly use organic fluorescent dyes and have weak fluorescent signals, and background noise brought by other biomolecules in the whole reaction system reduces the detection signal-to-noise ratio, so that the target nucleic acid fragment can only be amplified by various enzymes with different functions or hybridization methods, the concentration of the target nucleic acid fragment in the reaction system is increased, and further the signal is improved, so that the existence of the target nucleic acid fragment is obtained through scientific research and detection of instruments. However, both the traditional PCR amplification technology and the existing rapid amplification methods such as isothermal amplification and the like require complicated operations and short reaction time, and become the most difficult barrier for rapidly detecting nucleic acid molecules; therefore, a rapid and simple nucleic acid detection technology is of great importance and necessity for important work such as prevention and control of new infectious disease epidemic situations and biochemical safety in the future.

Disclosure of Invention

The invention aims to provide a rapid nucleic acid detection method based on an up-conversion fluorescent probe, aiming at the problems of complex operation and long reaction time of the existing nucleic acid molecule detection method.

The invention is realized by the following technical scheme: a nucleic acid rapid detection method based on an up-conversion fluorescent probe is used for detecting the number of nucleic acid molecules in a target sample and comprises the fluorescent probe obtained by modifying up-conversion fluorescent nanoparticles by a labeled nucleic acid fragment, a detection substrate modified by a capture nucleic acid fragment and the target sample; the target sample comprises target nucleic acid fragments, the capture nucleic acid fragments can be matched and hybridized with a part of the target nucleic acid fragments, and the marker nucleic acid fragments can be matched and hybridized with a part of the target nucleic acid fragments which cannot be matched and hybridized with the capture nucleic acid fragments;

the detection method comprises the following steps:

(1) diluting a target sample by using 0.2mol/L PB buffer solution to obtain an experimental standard substance with corresponding concentration, uniformly dripping the experimental standard substance onto a detection substrate, washing the detection substrate after the target sample is combined with a captured nucleic acid fragment on the detection substrate, and washing away the unbound target sample;

the preparation method of the target sample comprises the following steps: carrying out nucleic acid extraction on a target sample to be detected to obtain a target nucleic acid fragment, and dissociating the target nucleic acid fragment into a DNA single-stranded sample, wherein the DNA single-stranded sample is the target sample;

(2) diluting the fluorescent probe with 0.2mol/L PB buffer solution to obtain fluorescent probe diluent, uniformly dropwise adding the fluorescent probe diluent to the surface of the detection substrate treated in the step (1) for hybridization, washing the detection substrate, and washing away the unbound fluorescent probe;

(3) and (3) placing the detection substrate processed in the step (2) 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 fluorescent probes, wherein the obtained number is the number of the nucleic acid molecules in the target sample.

Specifically, the fluorescent probe is NaYF₄、NaGdF₄、CaF₂、LiYF₄、NaLuF₄、LiLuF₄、KMnF₃Or Y₂O₃The method comprises the steps of co-doping a luminescent substrate with a sensitizer and an activator to obtain up-conversion fluorescent nanoparticles UCNPs, then carrying out carboxylation modification on the surface of the UCNPs particles to obtain carboxylation modified nanoparticles UCNP @ PEG-COOH, and modifying the UCNP @ PEG-COOH after amination treatment of a labeled nucleic acid fragment to obtain a fluorescent probe;

in order to further explain the above, the method for preparing the fluorescent probe comprises:

(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⁺A sensitizer, an activator;

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 52-58 ℃ to obtain UCNPs;

(2) preparation of carboxyl modified nano-particles

Adding 1mL of 10mM UCNPs cyclohexane solution into 1mL of 0.3mol/L hydrochloric acid solution, performing ultrasonic treatment for 10min, stirring for 3h to obtain a mixed solution, centrifuging the mixed solution at a high speed, removing a supernatant to obtain a first precipitate, washing the first precipitate for 5-8 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 3-5 times to remove the redundant PEG-COOH, and obtaining UCNP @ PEG-COOH;

(3) fluorescent probe preparation

Dispersing UCNP @ PEG-COOH into 1mL of deionized water to obtain UCNP @ PEG-COOH dispersion liquid, adding the UCNP @ PEG-COOH dispersion liquid into 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 1-2mg/mL, then adding NHS with the concentration of 50mg/mL and EDC with the concentration of 50mg/mL into the suspension liquid in sequence to ensure that the mass ratio of the NHS to the luminescent substrate is 1:2-5 and the mass ratio of the EDC to the UCNPs is 1:2-5, then reacting for 20-60min at the temperature of 4-30 ℃, centrifuging to remove redundant NHS and EDC, then adding 0.2mol/L of PB buffer solution to obtain a second suspension liquid, adding an amination labeled nucleic acid fragment into the second suspension liquid, reacting for 1-3 hours at the temperature of 4-30 ℃, the mass ratio of the labeled nucleic acid fragment to the UCNPs is 1:100-1000, and BSA solution with the mass concentration of 0.5-2% is added for blocking for 1-2 hours after the reaction is finished; and after the reaction is finished, centrifugally washing for 1-2 times at the temperature of 3-5 ℃ to obtain a UCNPs-labeled nucleic acid fragment conjugate, namely the fluorescent probe, storing the UCNPs-labeled nucleic acid fragment conjugate in a PB buffer solution with the concentration of 0.02mol/L, and storing at the temperature of 2-8 ℃ for later use.

Specifically, the RECl3 solution RE³⁺The mol percentage of the mesosensitizer is 15mol%, 20mol%, 25mol%, 30mol%, 35mol%, 40mol%, 45mol%, 50mol%, 55mol%, 60mol%, 65mol%, 70mol% or 75 mol%; RECl3 solution RE³⁺The molar percentage of the medium activator is 2.5mol%, 3mol%, 4mol%, 5mol%, 6mol%, 7mol% or 8 mol%; the sensitizer is Yb³⁺、Nd³⁺、Gd³⁺、Ce³⁺One or more of them are mixed; the activator is Yb³⁺、Er³⁺、Tm³⁺、Sm³⁺、Dy³⁺、Ho³⁺、Eu³⁺、Tb³⁺、Pr³⁺One or more of them are mixed.

Specifically, the particle size of the up-conversion nanoparticles is 25-200 nm; the solute in the PB buffer comprises 0.1% BSA, 2% sucrose and 0.1% Tween according to mass percentage.

Specifically, when the target nucleic acid fragment is RNA, carrying out reverse transcription on the target nucleic acid fragment to obtain a DNA double strand, and then dissociating the DNA double strand into a DNA single strand sample; when the target nucleic acid fragment is a DNA double strand, the target nucleic acid fragment is dissociated into a DNA single strand sample.

Specifically, 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.

During detection, processing a target sample to obtain a DNA single-stranded sample, diluting an experimental standard substance by using a PB buffer solution, uniformly dripping the diluted experimental standard substance onto a detection substrate for a capture hybridization step, washing the detection substrate after completing pairing hybridization of a capture nucleic acid fragment and a part of a target nucleic acid fragment, then diluting a fluorescent probe, dripping the diluted fluorescent probe onto the detection substrate, and washing the detection substrate after completing pairing hybridization of a marker nucleic acid fragment on the fluorescent probe and the capture nucleic acid fragment; and (3) placing the washed 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 fluorescent probes, wherein the obtained number is the number of the nucleic acid molecules in the target sample.

Compared with the prior art, the invention has the following advantages: the rare earth-doped up-conversion fluorescent nano-particles UCNPs are used as the basis of a fluorescent probe, so that the UCNPs can be read under a fluorescent microscope; the corresponding capture nucleic acid fragment and the corresponding labeled nucleic acid fragment are designed according to the target nucleic acid fragment, the number of nucleic acid molecules in a target sample containing different target nucleic acid fragments can be detected, the application range is wide, the accuracy is high, compared with the traditional rare earth up-conversion fluorescent material, the single-molecule detection is realized, and the nucleic acid molecules can be observed by naked eyes without amplification and can be quantified.

Drawings

FIG. 1 is a schematic diagram of the structure of a fluorescent probe.

Fig. 2 is a schematic structural view of the detection substrate.

FIG. 3 is a schematic diagram of the tuberculosis reaction.

FIG. 4 is an illustration of the results of fluorescence microscopy images.

FIG. 5 is a quantitative chart of the detection results of HIV virus nucleic acid fragments.

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 rare earth doped up-conversion fluorescent nanoparticle is used as a luminescent material, the up-conversion nanoparticle can continuously absorb two or more long-wavelength photons and then emit a short-wavelength photon, so that the up-conversion fluorescent nanoparticle has larger anti-Stokes shift, 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, the signal to noise ratio of a detection signal can be obviously improved, the quantum efficiency is increased compared with the traditional rare earth doped up-conversion fluorescent material, the luminous intensity is greatly restricted, the single molecule detection level can be realized, the luminous intensity is higher, the signal of a single up-conversion luminescent probe can be observed by naked eyes, and the nucleic acid molecule can be observed and quantified without amplification.

As shown in fig. 1-2, a method for rapidly detecting nucleic acid based on an upconversion fluorescent probe, which is used for detecting the number of nucleic acid molecules in a target sample, comprises a fluorescent probe obtained by modifying an upconversion fluorescent nanoparticle 1 with a labeled nucleic acid fragment 2, a detection substrate 3 modified with a capture nucleic acid fragment 4, and a target sample; the target sample contains target nucleic acid fragments 5, as shown in FIG. 3, the capture nucleic acid fragments 4 can be paired and hybridized with a part of the target nucleic acid fragments 5, and the marker nucleic acid fragments 2 can be paired and hybridized with a part of the target nucleic acid fragments 5 which cannot be paired and hybridized with the capture nucleic acid fragments 4;

the detection method comprises the following steps:

(1) diluting a target sample by using 0.2mol/L PB buffer solution to obtain an experimental standard substance with corresponding concentration, uniformly dripping the experimental standard substance onto a detection substrate, washing the detection substrate after the target sample is combined with a captured nucleic acid fragment on the detection substrate, and washing away the unbound target sample;

the preparation method of the target sample comprises the following steps: carrying out nucleic acid extraction on a target sample to be detected to obtain a target nucleic acid fragment, and dissociating the target nucleic acid fragment into a DNA single-stranded sample, wherein the DNA single-stranded sample is the target sample;

(2) diluting the fluorescent probe with 0.2mol/L PB buffer solution to obtain fluorescent probe diluent, uniformly dropwise adding the fluorescent probe diluent to the surface of the detection substrate treated in the step (1) for hybridization, washing the detection substrate, and washing away the unbound fluorescent probe;

(3) and (3) placing the detection substrate processed in the step (2) 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 fluorescent probes, wherein the obtained number is the number of the nucleic acid molecules in the target sample.

Wherein the fluorescent probe is NaYF₄、NaGdF₄、CaF₂、LiYF₄、NaLuF₄、LiLuF₄、KMnF₃Or Y₂O₃The method comprises the steps of co-doping a luminescent substrate with a sensitizer and an activator to obtain up-conversion fluorescent nanoparticles UCNPs, then carrying out carboxylation modification on the surface of the UCNPs particles to obtain carboxylation modified nanoparticles UCNP @ PEG-COOH, and modifying the UCNP @ PEG-COOH after amination treatment of a labeled nucleic acid fragment to obtain a fluorescent probe;

the preparation method of the 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⁺A sensitizer, an activator;

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 52-58 ℃ to obtain UCNPs;

(2) preparation of carboxyl modified nano-particles

Adding 1mL of 10mM UCNPs cyclohexane solution into 1mL of 0.3mol/L hydrochloric acid solution, performing ultrasonic treatment for 10min, stirring for 3h to obtain a mixed solution, centrifuging the mixed solution at a high speed, removing a supernatant to obtain a first precipitate, washing the first precipitate for 5-8 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 3-5 times to remove the redundant PEG-COOH, and obtaining UCNP @ PEG-COOH;

(3) fluorescent probe preparation

Dispersing UCNP @ PEG-COOH into 1mL of deionized water to obtain UCNP @ PEG-COOH dispersion liquid, adding the UCNP @ PEG-COOH dispersion liquid into 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 1-2mg/mL, then adding NHS with the concentration of 50mg/mL and EDC with the concentration of 50mg/mL into the suspension liquid in sequence to ensure that the mass ratio of the NHS to the luminescent substrate is 1:2-5 and the mass ratio of the EDC to the UCNPs is 1:2-5, then reacting for 20-60min at the temperature of 4-30 ℃, centrifuging to remove redundant NHS and EDC, then adding 0.2mol/L of PB buffer solution to obtain a second suspension liquid, adding an amination labeled nucleic acid fragment into the second suspension liquid, reacting for 1-3 hours at the temperature of 4-30 ℃, the mass ratio of the labeled nucleic acid fragment to the UCNPs is 1:100-1000, and BSA solution with the mass concentration of 0.5-2% is added for blocking for 1-2 hours after the reaction is finished; and after the reaction is finished, centrifugally washing for 1-2 times at the temperature of 3-5 ℃ to obtain a UCNPs-labeled nucleic acid fragment conjugate, namely the fluorescent probe, storing the UCNPs-labeled nucleic acid fragment conjugate in a PB buffer solution with the concentration of 0.02mol/L, and storing at the temperature of 2-8 ℃ for later use.

Wherein the RECl3 solution RE³⁺The mol percentage of the mesosensitizer is 15mol%, 20mol%, 25mol%, 30mol%, 35mol%, 40mol%, 45mol%, 50mol%, 55mol%, 60mol%, 65mol%, 70mol% or 75 mol%; RECl3 solution RE³⁺The molar percentage of the medium activator is 2.5mol%, 3mol%, 4mol%, 5mol%, 6mol%, 7mol% or 8 mol%; the sensitizer is Yb³⁺、Nd³⁺、Gd³⁺、Ce³⁺One or more of them are mixed; the activator is Yb³⁺、Er³⁺、Tm³⁺、Sm³⁺、Dy³⁺、Ho³⁺、Eu³⁺、Tb³⁺、Pr³⁺One or more of them are mixed; the particle size of the up-conversion nanoparticles is 25-200 nm; the solute in the PB buffer comprises 0.1% BSA, 2% sucrose and 0.1% Tween according to mass percentage.

Wherein 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.