Nanoprobe for rapidly detecting procalcitonin, preparation method of nanoprobe and detection method of procalcitonin
By designing a dual fluorescent probe modified by antibody, the FRET method is used to achieve high sensitivity detection of procalcitonin, which solves the problems of long detection time and insufficient sensitivity in the prior art, and provides a faster and more accurate detection method.
Patent Information
- Application Number
- CN202411576659.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-06
- Publication Date
- 2025-02-07
AI Technical Summary
The prior art has problems such as long detection time and insufficient sensitivity in procalcitonin detection, which is difficult to meet the clinical needs for rapid and accurate detection.
Using antibody-modified dual fluorescence probes, the donor probe B-CDs-Ab1 and the acceptor probe G-CDs-Ab2 were designed to achieve high procalcitonin by time-resolved fluorescence resonance energy transfer (FRET) method. Sensitivity-specific capture recognition.
It realizes rapid detection of procalcitonin, improves the sensitivity and accuracy of the detection, and provides more efficient detection methods for clinical practice.
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Figure CN119394990A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the fields of medicine and nanomaterials, and in particular to a nanoprobe for rapid detection of procalcitonin, a preparation method thereof and a method for detecting procalcitonin. Background Art
[0002] The rational use of antibiotics is the core of the treatment of bacterial infectious diseases. Long-term antibiotic treatment is one of the main causes of drug resistance, and the appropriate timing of drug withdrawal is still a huge challenge in clinical practice. The rapid development of laboratory diagnostic technology has provided more references for the rational use of antibiotics.
[0003] Procalcitonin (PCT) has been widely used as an important reference indicator for the diagnosis and treatment of bacterial infectious diseases. The guiding value of PCT in the course of antimicrobial treatment for patients with lower respiratory tract infections and severe infections in intensive care units (ICUs) has gradually been recognized. In recent years, PCT has been considered to be a specific indicator of the body's systemic inflammatory response to bacterial infection and contains 116 amino acids. Sepsis is a daily challenge faced by hospitals, with a mortality rate of up to 50%. Various treatment strategies are currently known to improve the survival rate of patients with sepsis. The earlier sepsis is identified and treated, the better the prognosis. Acute respiratory tract infection is the main cause of sepsis, accounting for about 10% of global morbidity and mortality. Under normal physiological conditions, PCT is only synthesized by thyroid C cells and is present in very low levels in plasma. In the case of systemic inflammatory response and sepsis, PCT can be produced in large quantities by tissues other than the thyroid gland, and the PCT content in plasma increases rapidly. PCT has a half-life of 25 to 30 hours in the blood, good in vivo stability, and no significant difference in the detection values in serum and plasma. It has high clinical value for early diagnosis of bacterial infection or sepsis, improving diagnostic accuracy, judging the severity of infectious diseases, and evaluating the prognosis of infectious diseases. In recent years, traditional quantitative and semi-quantitative detection methods including colloidal gold labeling, radioimmunoassay, immunofluorescence, double antibody sandwich immunochemiluminescence, and enzyme immunoassay have played an important role in PCT detection, but they have certain limitations, including long consumption time and insufficient test sensitivity. Therefore, faster and more accurate detection technologies are needed to make up for these shortcomings and provide more efficient detection means and guarantees for clinical detection.
[0004] The time-resolved fluorescence resonance energy transfer (FRET) method combines fluorescent nanomaterials and modified antibodies to form a dual fluorescent probe. The donor emission spectrum and the acceptor excitation spectrum are effectively overlapped to achieve fluorescence energy transfer, prolong the reaction time and facilitate observation, especially in high-sensitivity detection projects with potential application prospects. It effectively couples the functional groups on the surface of two fluorescent nanomaterials with the recognition antibody end, forms an "antibody-antigen-antibody" according to the "double antibody sandwich method", quickly realizes donor-acceptor energy transfer, and realizes high-sensitivity detection of molecules. Therefore, the use of antibody-modified fluorescent nanomaterials to prepare probes to realize the FRET method of resonance energy transfer provides a new method and idea for the rapid detection of clinical high-sensitivity projects. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a nanoprobe for rapid detection of procalcitonin, a preparation method thereof and a method for detecting procalcitonin in view of the deficiencies in the above-mentioned prior art.
[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: In a first aspect of the present invention, a method for preparing a nanoprobe for rapid detection of procalcitonin is provided, wherein the nanoprobe comprises a donor probe B-CDs-Ab1 and an acceptor probe G-CDs-Ab2, and the method comprises the following steps:
[0007] S1. Synthesis of donor fluorescent nanoparticles B-CDs:
[0008] S1-1, dissolving citric acid powder in purified water under magnetic stirring until the solution becomes transparent; then, adding ethylenediamine to form a uniform mixed solution 1;
[0009] S1-2, adding the mixed solution 1 to a high-pressure reactor, reacting under heating, cooling to room temperature after the reaction is completed, filtering the obtained product solution, mixing the obtained filtrate with silica gel, and evaporating it using a rotary evaporator to obtain a crude product powder 1;
[0010] S1-3, purifying the crude product powder 1 by a chromatographic column separation method, using a mixture of methanol and dichloromethane as an eluent for gradient elution, mixing the collected elution solution with silica gel for a second spin evaporation, and finally obtaining donor fluorescent nanoparticles: B-CDs, which are stored for later use;
[0011] S2. Synthesis of acceptor fluorescent nanoparticles G-CDs:
[0012] dissolving passionflower extract powder and m-phenylenediamine powder in purified water under magnetic stirring to obtain a mixed solution 2;
[0013] The mixed solution 2 was added to a high-pressure reactor, and reacted under heating. After the reaction was completed, the mixture was cooled to room temperature, and the obtained product solution was centrifuged. The supernatant obtained by centrifugation was mixed with silica gel and then evaporated using a rotary evaporator to obtain a crude product powder 2;
[0014] The crude product powder 2 is purified by a chromatographic column separation method, and a mixture of methanol and dichloromethane is used as an eluent for gradient elution. The collected elution solution is mixed with silica gel and subjected to a second spin evaporation, and finally the acceptor fluorescent nanoparticles: G-CDs are obtained, which are stored for later use;
[0015] S3. Synthesize donor probe B-CDs-Ab1:
[0016] S3-1, dissolving the donor fluorescent nanoparticles B-CDs in a PBS solution, filtering and discarding the residue to obtain a B-CDs solution;
[0017] S3-2, add EDC and NHS to the B-CDs solution, incubate, then add the antibody 1 solution and incubate;
[0018] Wherein, the antibody 1 solution is the Ab1 solution prepared with PBS;
[0019] S3-3, centrifuge the product obtained in step S3-2, discard the supernatant to obtain B-CDs-Ab1 concentrate, add PBS buffer solution, centrifuge and wash again to remove EDC, NHS and antibody 1 that have not participated in the reaction, and obtain the donor probe: B-CDs-Ab1, which is stored for future use;
[0020] S4. Synthetic receptor probe G-CDs-Ab2:
[0021] S4-1, dissolving the acceptor fluorescent nanoparticles G-CDs in a PBS solution, filtering, and discarding the residue to obtain a G-CDs solution;
[0022] S4-2, adding EDC and NHS to the G-CDs solution, incubating, and then adding the antibody 2 solution, incubating;
[0023] Wherein, the antibody 2 solution is the Ab2 solution prepared with PBS;
[0024] S4-3. Centrifuge the product obtained in step S4-2, retain the supernatant to obtain B-CDs-Ab1 concentrate, add PBS buffer solution and centrifuge again to remove EDC, NHS and antibody 2 that have not participated in the reaction, and obtain the receptor probe: G-CDs-Ab2, which is stored for later use.
[0025] Preferably, step S1 specifically comprises:
[0026] S1-1, dissolve 0.5-2 g of citric acid powder in 5-30 mL of purified water under magnetic stirring until the solution becomes transparent; then, add 150-600 μL of ethylenediamine to form a uniform mixed solution 1;
[0027] S1-2, adding the mixed solution 1 into a high pressure reactor, reacting at 160-200°C for 3-12 hours, cooling to room temperature after the reaction, filtering the obtained product solution using a 0.2-0.3 μm microporous membrane, mixing the obtained filtrate with 1-4 g of silica gel, and evaporating using a rotary evaporator to obtain a crude product powder 1;
[0028] S1-3. Purify the crude product powder 1 by chromatographic column separation method, use a mixture of methanol and dichloromethane in a volume ratio of 1:10 as an eluent for gradient elution, mix the collected elution solution with 1-4g silica gel and perform a second spin evaporation to finally obtain donor fluorescent nanoparticles: B-CDs, which are stored at 0-5°C for future use.
[0029] Preferably, step S2 specifically comprises:
[0030] S2. Synthesis of acceptor fluorescent nanoparticles G-CDs:
[0031] Dissolve 0.25-1 g of passionflower extract powder and 0.4-1.6 g of m-phenylenediamine powder in 15-60 mL of purified water under magnetic stirring to obtain a mixed solution 2;
[0032] The mixed solution 2 is added to a high-pressure reactor, and reacted at 140-180° C. for 4-16 hours. After the reaction is completed, the mixture is cooled to room temperature. The obtained product solution is centrifuged at 5000-20000 rpm for 15-60 minutes. The supernatant obtained by centrifugation is mixed evenly with 1-4 g of silica gel and then evaporated using a rotary evaporator to obtain a crude product powder 2.
[0033] The crude product powder 1 was purified by a chromatographic column separation method, and a mixture of methanol and dichloromethane in a volume ratio of 1:10 was used as an eluent for gradient elution. The collected elution solution was mixed with 1-4 g of silica gel and subjected to a second spin evaporation to finally obtain receptor fluorescent nanoparticles: G-CDs, which were stored at 0-5°C for future use.
[0034] Preferably, step S3 specifically comprises:
[0035] S3. Synthesize donor probe B-CDs-Ab1:
[0036] S3-1, take 0.05-0.2g of donor fluorescent nanoparticles B-CDs and dissolve it in 5-20mL of 0.01M PBS solution, filter it using a 0.2-0.3μm microporous membrane, discard the filter residue, and obtain a B-CDs solution;
[0037] S3-2, add 10-40 mg EDC and 5-20 mg NHS to 0.5-2 mL B-CDs solution, shake and incubate for 15-60 min at room temperature on a shaker, then add 12.5-50 μL antibody 1 solution to the B-CDs solution, and incubate for 2-8 hours on a shaker at room temperature;
[0038] The antibody 1 solution is an Ab1 solution with a concentration of 0.25-1 mg / mL prepared with 0.01 M PBS;
[0039] S3-3. Take 150-600 μL of the product obtained in step S3-2 and add it to an ultrafiltration centrifuge tube. Centrifuge at 5000-20000 rpm for 30-90 min. Keep the supernatant to obtain B-CDs-Ab1 concentrate. Add 150-600 μL of PBS buffer solution and centrifuge again to remove EDC, NHS and antibody 1 that have not participated in the reaction to obtain the donor probe: B-CDs-Ab1. Store at 0-5°C for future use.
[0040] Preferably, step S4 is specifically:
[0041] S4. Synthetic receptor probe G-CDs-Ab2:
[0042] S4-1, taking 0.05-0.2g of acceptor fluorescent nanoparticles G-CDs and dissolving them in 5-20mL 0.01M PBS solution, filtering them through a 0.2-0.3μm microporous membrane, discarding the filter residue, and obtaining a G-CDs solution;
[0043] S4-2, add 10-40 mg EDC and 5-20 mg NHS to 0.5-2 mL G-CDs solution, shake and incubate for 15-60 min at room temperature on a shaker, then add 12.5-50 μL antibody 2 solution to the G-CDs solution, and incubate at 0-5°C on a shaker for 10-40 hours;
[0044] The antibody 2 solution is an Ab2 solution with a concentration of 0.25-1 mg / mL prepared with 0.01 M PBS;
[0045] S4-3. Take 150-600 μL of the product obtained in step S4-2 and add it to an ultrafiltration centrifuge tube. Centrifuge at 5000-20000 rpm for 30-90 min. Keep the supernatant to obtain B-CDs-Ab1 concentrate. Add 150-600 μL of PBS buffer solution and centrifuge again to remove EDC, NHS and antibody 2 that have not participated in the reaction to obtain the receptor probe: G-CDs-Ab2. Store at 0-5°C for later use.
[0046] Preferably, the method for preparing the nanoprobe for rapid detection of procalcitonin comprises the following steps:
[0047] S1. Synthesis of donor fluorescent nanoparticles B-CDs:
[0048] S1-1, dissolve 1.051 g of citric acid powder in 15 mL of purified water under magnetic stirring until the solution becomes transparent; then, add 335 μL of ethylenediamine to form a uniform mixed solution 1;
[0049] S1-2, adding the mixed solution 1 into a high-pressure reactor, reacting at 180° C. for 6 h, cooling to room temperature after the reaction, filtering the obtained product solution using a 0.22 μm microporous membrane, mixing the obtained filtrate with 2 g of silica gel, and evaporating using a rotary evaporator to obtain a crude product powder 1;
[0050] S1-3. Purify the crude product powder 1 by a chromatographic column separation method, use a mixture of methanol and dichloromethane in a volume ratio of 1:10 as an eluent for gradient elution, mix the collected elution solution with 2g of silica gel and perform a second spin evaporation, and finally obtain donor fluorescent nanoparticles: B-CDs, which are stored at 4°C for future use;
[0051] S2. Synthesis of acceptor fluorescent nanoparticles G-CDs:
[0052] 0.5 g of passionflower extract powder and 0.8 g of m-phenylenediamine powder were dissolved in 30 mL of purified water under magnetic stirring to obtain a mixed solution 2;
[0053] The mixed solution 2 was added to a high-pressure reactor and reacted at 160° C. for 8 h. After the reaction was completed, the mixture was cooled to room temperature. The obtained product solution was centrifuged at 10,000 rpm for 30 min. The supernatant obtained by centrifugation was mixed with 2 g of silica gel and then evaporated using a rotary evaporator to obtain a crude product powder 2.
[0054] The crude product powder 1 was purified by chromatographic column separation method, and a mixture of methanol and dichloromethane in a volume ratio of 1:10 was used as an eluent for gradient elution. The collected elution solution was mixed with 2g of silica gel for a second spin evaporation, and finally the receptor fluorescent nanoparticles: G-CDs were obtained, which were stored at 4°C for future use;
[0055] S3. Synthesize donor probe B-CDs-Ab1:
[0056] S3-1, take 0.1g of donor fluorescent nanoparticles B-CDs and dissolve it in 10mL of 0.01M PBS solution, filter it using a 0.22μm microporous membrane, discard the filter residue, and obtain a B-CDs solution;
[0057] S3-2, add 20 mg EDC and 10 mg NHS to 1 mL B-CDs solution, shake and incubate for 30 min at room temperature on a shaker, then add 25 μL antibody 1 solution to the B-CDs solution, and incubate for 4 h at room temperature on a shaker;
[0058] The antibody 1 solution is an Ab1 solution with a concentration of 0.5 mg / mL prepared with 0.01 M PBS. More preferably, the Ab1 antibody is V0309, purchased from Beijing Bioson Biotechnology Co., Ltd.;
[0059] S3-3. Take 300 μL of the product obtained in step S3-2 and add it to an ultrafiltration centrifuge tube. Centrifuge at 10,000 rpm for 45 min. Keep the supernatant to obtain B-CDs-Ab1 concentrate. Add 300 μL of PBS buffer solution and centrifuge again to remove EDC, NHS and antibody 1 that have not participated in the reaction to obtain the donor probe: B-CDs-Ab1. Store at 4°C for future use.
[0060] S4. Synthetic receptor probe G-CDs-Ab2:
[0061] S4-1, take 0.1g of acceptor fluorescent nanoparticles G-CDs and dissolve it in 10mL of 0.01M PBS solution, filter it using a 0.22μm microporous membrane, discard the filter residue, and obtain a G-CDs solution;
[0062] S4-2, add 20 mg EDC and 10 mg NHS to 1 mL G-CDs solution, shake and incubate for 30 min at room temperature on a shaker, then add 25 μL antibody 2 solution to the G-CDs solution and incubate for 20 h on a shaker at 4 °C;
[0063] The antibody 2 solution is an Ab2 solution with a concentration of 0.5 mg / mL prepared with 0.01 M PBS. More preferably, the Ab2 antibody is V0306, purchased from Beijing Bioson Biotechnology Co., Ltd.;
[0064] S4-3. Take 300 μL of the product obtained in step S4-2 and add it to an ultrafiltration centrifuge tube. Centrifuge at 10,000 rpm for 45 min. Keep the supernatant to obtain B-CDs-Ab1 concentrate. Add 300 μL of PBS buffer solution and centrifuge again to remove EDC, NHS and antibody 2 that have not participated in the reaction to obtain the receptor probe: G-CDs-Ab2. Store at 4°C for future use.
[0065] The second aspect of the present invention provides a nanoprobe for rapid detection of procalcitonin, characterized in that it is prepared by the method as described above.
[0066] The third aspect of the present invention provides a method for rapid detection of procalcitonin, which uses the nanoprobe as described above for detection, and the method is:
[0067] The donor probe and the acceptor probe are mixed with the test solution containing PCT antigen, incubated, and the fluorescence intensity of the emission light of the reaction product under the excitation light at 450nm and 505nm is detected, which are recorded as F 450 and F 505 , calculate F 505 / F 450 The value of F was then calculated based on the pre-established relationship between PCT antigen concentration and F 505 / F 450 The PCT antigen concentration in the test solution is calculated based on the standard curve of the relationship between the values of
[0068] Preferably, the method for rapid detection of procalcitonin is:
[0069] The donor probe, the acceptor probe and the test solution containing PCT antigen were mixed and incubated at 37°C for 30 min. The fluorescence intensity of the reaction product under the excitation light of 464 nm, the emission light at 450 nm and 505 nm were detected, which were recorded as F 450 and F 505 , calculate F 505 / F 450 The value of F was then calculated based on the pre-established relationship between PCT antigen concentration and F 505 / F 450 The PCT antigen concentration in the test solution is calculated based on the standard curve of the relationship between the values of
[0070] Preferably, the method for constructing a standard curve comprises the following steps:
[0071] 1) using antigen-antibody diluent and PCT antigen to prepare a series of working solutions containing different PCT antigen concentrations;
[0072] 2) adding the donor probe and the acceptor probe to the antigen-antibody dilution solution to obtain a probe solution, and preparing several equal volumes of the probe solution;
[0073] 3) Add the same volume of working solution with different concentrations to each probe solution, incubate the resulting mixed solution, and detect the fluorescence intensity of the emission light of the reaction product under the excitation light at 450nm and 505nm, which are recorded as F 450 and F 505 , with PCT antigen concentration as the horizontal axis, F 505 / F 450 The value is the ordinate, and the fitting results are used to characterize the relationship between PCT antigen concentration and F 505 / F 450 The standard curve of the relationship between the values of
[0074] The antigen-antibody diluent is a PBS solution containing BSA.
[0075] Preferably, the method for constructing a standard curve comprises the following steps:
[0076] 1) Using antigen-antibody diluent and PCT antigen, prepare working solutions with PCT antigen concentrations of 0, 0.1, 0.5, 1, 5, 10, 20, 40, 60, and 80 ng / mL, respectively;
[0077] 2) Add 20 μL of donor probe and 20 μL of acceptor probe to 180 μL of antigen-antibody diluent to obtain a probe solution, and prepare several aliquots of the probe solution;
[0078] 3) 100 μL of working solution of different concentrations was added to each probe solution, and the resulting mixed solution was incubated at 37°C for 30 min. The fluorescence intensity of the reaction product under the excitation light of 464 nm, the emission light at 450 nm and 505 nm was detected, which were recorded as F 450 and F 505 , with PCT antigen concentration as the horizontal axis, F 505 / F 450 The value is the ordinate, and the fitting results are used to characterize the relationship between PCT antigen concentration and F 505 / F 450 The standard curve of the relationship between the values of
[0079] The antigen-antibody diluent is a PBS solution containing 3 wt % BSA at a concentration of 0.01 M.
[0080] The beneficial effects of the present invention are:
[0081] The present invention provides a nanoprobe for rapid detection of procalcitonin, a preparation method thereof, and a procalcitonin detection method based on the nanoprobe. The present invention achieves high-sensitivity and specific capture and recognition of procalcitonin by designing and preparing an antibody-modified dual probe. Based on the expression of different fluorescent signal molecules of donors and acceptors, rapid detection of procalcitonin can be achieved, thereby providing a new tool for high-sensitivity rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0082] Figure 1 TEM photos of B-CDs and G-CDs prepared in Example 1 of the present invention;
[0083] Figure 2 The infrared spectra of B-CDs and B-CDs-Ab1, G-CDs and G-CDs-Ab2 prepared in Example 1 of the present invention;
[0084] Figure 3The fluorescence spectra of B-CDs-Ab1 and G-CDs-Ab2 prepared in Example 1 of the present invention detecting antigens at different concentrations;
[0085] Figure 4 This is the standard curve constructed in Example 2 of the present invention. DETAILED DESCRIPTION
[0086] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.
[0087] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.
[0088] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.
[0089] Example 1
[0090] A nanoprobe for rapid detection of procalcitonin, the nanoprobe comprising a donor probe B-CDs-Ab1 and an acceptor probe G-CDs-Ab2, and a preparation method thereof comprising the following steps:
[0091] S1. Synthesis of donor fluorescent nanoparticles B-CDs:
[0092] S1-1, dissolve 1.051 g of citric acid powder in 15 mL of purified water under magnetic stirring until the solution becomes transparent; then, add 335 μL of ethylenediamine to form a uniform mixed solution 1;
[0093] S1-2, adding the mixed solution 1 into a high-pressure reactor, reacting at 180° C. for 6 h, cooling to room temperature after the reaction, filtering the obtained product solution using a 0.22 μm microporous membrane, mixing the obtained filtrate with 2 g of silica gel, and evaporating using a rotary evaporator to obtain a crude product powder 1;
[0094] S1-3. Purify the crude product powder 1 by a chromatographic column separation method (dry loading), use a mixture of methanol and dichloromethane in a volume ratio of 1:10 as an eluent for gradient elution, mix the collected elution solution with 2 g of silica gel and perform a second spin evaporation, and finally obtain donor fluorescent nanoparticles with blue emission fluorescence: B-CDs, which are stored at 4°C for future use;
[0095] S2. Synthesis of acceptor fluorescent nanoparticles G-CDs:
[0096] 0.5 g of passionflower extract powder and 0.8 g of m-phenylenediamine powder were dissolved in 30 mL of purified water under magnetic stirring to obtain a mixed solution 2;
[0097] The mixed solution 2 was added to a high-pressure reactor and reacted at 160° C. for 8 h. After the reaction was completed, the mixture was cooled to room temperature. The obtained product solution was centrifuged at 10,000 rpm for 30 min. The supernatant obtained by centrifugation was mixed with 2 g of silica gel and then evaporated using a rotary evaporator to obtain a crude product powder 2.
[0098] The crude product powder 1 was purified by a chromatographic column separation method (dry loading), and a mixture of methanol and dichloromethane in a volume ratio of 1:10 was used as an eluent for gradient elution. The collected elution solution was mixed with 2g of silica gel for a second spin evaporation, and finally acceptor fluorescent nanoparticles with green emission fluorescence: G-CDs were obtained, which were stored at 4°C for future use;
[0099] S3. Synthesize donor probe B-CDs-Ab1:
[0100] S3-1, take 0.1g of donor fluorescent nanoparticles B-CDs and dissolve it in 10mL of 0.01M PBS solution, filter it using a 0.22μm microporous membrane, discard the filter residue, and obtain a B-CDs solution;
[0101] S3-2, add 20 mg EDC and 10 mg NHS to 1 mL B-CDs solution, shake and incubate for 30 min at room temperature on a shaker, then add 25 μL antibody 1 solution to the B-CDs solution, and incubate for 4 h at room temperature on a shaker;
[0102] The antibody 1 solution was a 0.5 mg / mL Ab1 solution prepared with 0.01 M PBS, and the Ab1 antibody was V0309, purchased from Beijing Bioson Biotechnology Co., Ltd.;
[0103] S3-3. Take 300 μL of the product obtained in step S3-2 and add it to an ultrafiltration centrifuge tube. Centrifuge at 10,000 rpm for 45 min. Keep the supernatant to obtain B-CDs-Ab1 concentrate. Add 300 μL of PBS buffer solution and centrifuge again to remove EDC, NHS and antibody 1 that have not participated in the reaction to obtain the donor probe: B-CDs-Ab1. Store at 4°C for future use.
[0104] S4. Synthetic receptor probe G-CDs-Ab2:
[0105] S4-1, take 0.1g of acceptor fluorescent nanoparticles G-CDs and dissolve it in 10mL of 0.01M PBS solution, filter it using a 0.22μm microporous membrane, discard the filter residue, and obtain a G-CDs solution;
[0106] S4-2, add 20 mg EDC and 10 mg NHS to 1 mL G-CDs solution, shake and incubate for 30 min at room temperature on a shaker, then add 25 μL antibody 2 solution to the G-CDs solution and incubate for 20 h on a shaker at 4 °C;
[0107] The antibody 2 solution was an Ab2 solution with a concentration of 0.5 mg / mL prepared with 0.01 M PBS, and the Ab2 antibody was V0306, purchased from Beijing Bioson Biotechnology Co., Ltd.;
[0108] S4-3. Take 300 μL of the product obtained in step S4-2 and add it to an ultrafiltration centrifuge tube. Centrifuge at 10,000 rpm for 45 min. Keep the supernatant to obtain B-CDs-Ab1 concentrate. Add 300 μL of PBS buffer solution and centrifuge again to remove EDC, NHS and antibody 2 that have not participated in the reaction to obtain the receptor probe: G-CDs-Ab2. Store at 4°C for future use.
[0109] Reference Figure 1 , which are transmission electron microscope (TEM) photos of B-CDs (A) and G-CDs (B) prepared in Example 1. It can be seen from the figure that the size distribution of the nanoparticles is relatively uniform. It is observed that the nanoparticles are in a regular spherical shape, and the average particle size is about 2-8nm.
[0110] Reference Figure 2 , which are the infrared spectra of B-CDs and the donor probe B-CDs-Ab1 of the surface amino-coupled antibody (A); and the infrared spectra of G-CDs and the acceptor probe G-CDs-Ab2 of the surface amino-coupled antibody (B).
[0111] Example 2
[0112] A method for rapid detection of procalcitonin, the method using the nanoprobe of Example 1 for detection, the method comprising:
[0113] The donor probe, the acceptor probe and the test solution containing PCT antigen were mixed and incubated at 37°C for 30 min. The fluorescence intensity of the reaction product under the excitation light of 464 nm, the emission light at 450 nm and 505 nm were detected, which were recorded as F 450 and F 505 , calculate F 505 / F 450 The value of F was then calculated based on the pre-established relationship between PCT antigen concentration and F 505 / F 450 The PCT antigen concentration in the test solution is calculated based on the standard curve of the relationship between the values of
[0114] In this embodiment, the method for constructing a standard curve includes the following steps:
[0115] 1) Using antigen-antibody diluent and PCT antigen, prepare working solutions with PCT antigen concentrations of 0, 0.1, 0.5, 1, 5, 10, 20, 40, 60, and 80 ng / mL, respectively;
[0116] 2) Add 20 μL of donor probe and 20 μL of acceptor probe to 180 μL of antigen-antibody diluent to obtain a probe solution, and prepare several aliquots of the probe solution;
[0117] 3) 100 μL of working solution of different concentrations was added to each probe solution, and the resulting mixed solution was incubated at 37°C for 30 min. The fluorescence intensity of the reaction product under the excitation light of 464 nm, the emission light at 450 nm and 505 nm was detected, which were recorded as F 450 and F 505 , with PCT antigen concentration as the horizontal axis, F 505 / F 450 The value is the ordinate, and the fitting results are used to characterize the relationship between PCT antigen concentration and F 505 / F 450 The standard curve of the relationship between the values of
[0118] The antigen-antibody diluent is a PBS solution containing 3 wt % BSA at a concentration of 0.01 M.
[0119] Reference Figure 3 During the construction of the standard curve, the fluorescence intensity changes of the nanoprobes: B-CDs-Ab1 and G-CDs-Ab2 when detecting different concentrations of antigen. The degree of reaction between different concentrations of antigen and antibody is different. The higher the concentration, the faster the fluorescence energy transfer from the donor probe to the acceptor probe. The fluorescence change ratio of the donor and acceptor probes is proportional to the concentration of the detected antigen.
[0120] Reference Figure 4 , is the standard curve obtained by fitting. It can be seen that the linear curves corresponding to different antigen concentrations are different. The linear relationship of 0-5 ng / mL antigen concentration is a quadratic function.
[0121] F 505 / F 450 =-0.0045×C PCT 2 +0.0021×C PCT +0.3232, R 2=99.41%; the linear relationship of 5-80ng / mL antigen concentration is a linear function F 505 / F 450 =0.0021×C PCT +0.4212, R 2 =99.37%. Low concentration antigen detection is more sensitive, indicating that this method can be used in clinical testing for high-sensitivity trace detection.
[0122] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.
Claims
1. A method for preparing a nanoprobe for rapid detection of procalcitonin, characterized in that: The nanoprobe comprises a donor probe B-CDs-Ab1 and an acceptor probe G-CDs-Ab2, and the method comprises the following steps: S1. Synthesis of donor fluorescent nanoparticles B-CDs: S1-1, dissolving citric acid powder in purified water under magnetic stirring until the solution becomes transparent; then, adding ethylenediamine to form a uniform mixed solution 1; S1-2, adding the mixed solution 1 to a high-pressure reactor, reacting under heating, cooling to room temperature after the reaction is completed, filtering the obtained product solution, mixing the obtained filtrate with silica gel, and evaporating it using a rotary evaporator to obtain a crude product powder 1; S1-3, purifying the crude product powder 1 by a chromatographic column separation method, using a mixture of methanol and dichloromethane as an eluent for gradient elution, mixing the collected elution solution with silica gel for a second spin evaporation, and finally obtaining donor fluorescent nanoparticles: B-CDs, which are stored for later use; S2. Synthesis of acceptor fluorescent nanoparticles G-CDs: dissolving passionflower extract powder and m-phenylenediamine powder in purified water under magnetic stirring to obtain a mixed solution 2; The mixed solution 2 was added to a high-pressure reactor, and reacted under heating. After the reaction was completed, the mixture was cooled to room temperature, and the obtained product solution was centrifuged. The supernatant obtained by centrifugation was mixed with silica gel and then evaporated using a rotary evaporator to obtain a crude product powder 2; The crude product powder 2 was purified by a chromatographic column separation method, and a mixture of methanol and dichloromethane was used as an eluent for gradient elution. The collected elution solution was mixed with silica gel for a second spin evaporation, and finally the receptor fluorescent nanoparticles: G-CDs were obtained, which were stored for future use; S3. Synthesize donor probe B-CDs-Ab1: S3-1, dissolving the donor fluorescent nanoparticles B-CDs in a PBS solution, filtering and discarding the residue to obtain a B-CDs solution; S3-2, add EDC and NHS to the B-CDs solution, incubate, then add the antibody 1 solution and incubate; Wherein, the antibody 1 solution is the Ab1 solution prepared with PBS; S3-3, centrifuge the product obtained in step S3-2, discard the supernatant to obtain B-CDs-Ab1 concentrate, add PBS buffer solution, centrifuge and wash again to remove EDC, NHS and antibody 1 that have not participated in the reaction, and obtain the donor probe: B-CDs-Ab1, which is stored for future use; S4. Synthetic receptor probe G-CDs-Ab2: S4-1, dissolving the acceptor fluorescent nanoparticles G-CDs in a PBS solution, filtering, and discarding the residue to obtain a G-CDs solution; S4-2, adding EDC and NHS to the G-CDs solution, incubating, and then adding the antibody 2 solution, incubating; Wherein, the antibody 2 solution is the Ab2 solution prepared with PBS; S4-3. Centrifuge the product obtained in step S4-2, discard the supernatant, obtain B-CDs-Ab1 concentrate, add PBS buffer solution, centrifuge and wash again to remove EDC, NHS and antibody 2 that have not participated in the reaction, and obtain the receptor probe: G-CDs-Ab2, which is stored for later use.
2. The method for preparing a nanoprobe for rapid detection of procalcitonin according to claim 1, characterized in that: Step S1 is specifically as follows: S1-1, dissolve 0.5-2 g of citric acid powder in 5-30 mL of purified water under magnetic stirring until the solution becomes transparent; then, add 150-600 μL of ethylenediamine to form a uniform mixed solution 1; S1-2, adding the mixed solution 1 into a high pressure reactor, reacting at 160-200°C for 3-12 hours, cooling to room temperature after the reaction, filtering the obtained product solution using a 0.2-0.3 μm microporous membrane, mixing the obtained filtrate with 1-4 g of silica gel, and evaporating using a rotary evaporator to obtain a crude product powder 1; S1-3. Purify the crude product powder 1 by chromatographic column separation method, use a mixture of methanol and dichloromethane in a volume ratio of 1:10 as an eluent for gradient elution, mix the collected elution solution with 1-4g silica gel and perform a second spin evaporation to finally obtain donor fluorescent nanoparticles: B-CDs, which are stored at 0-5°C for future use.
3. The method for preparing a nanoprobe for rapid detection of procalcitonin according to claim 2, characterized in that: Step S2 is specifically as follows: S2. Synthesis of acceptor fluorescent nanoparticles G-CDs: Dissolve 0.25-1 g of passionflower extract powder and 0.4-1.6 g of m-phenylenediamine powder in 15-60 mL of purified water under magnetic stirring to obtain a mixed solution 2; The mixed solution 2 is added to a high-pressure reactor, and reacted at 140-180° C. for 4-16 hours. After the reaction is completed, the mixture is cooled to room temperature. The obtained product solution is centrifuged at 5000-20000 rpm for 15-60 minutes. The supernatant obtained by centrifugation is mixed evenly with 1-4 g of silica gel and then evaporated using a rotary evaporator to obtain a crude product powder 2. The crude product powder 1 was purified by a chromatographic column separation method, and a mixture of methanol and dichloromethane in a volume ratio of 1:10 was used as an eluent for gradient elution. The collected elution solution was mixed with 1-4 g of silica gel and subjected to a second spin evaporation to finally obtain receptor fluorescent nanoparticles: G-CDs, which were stored at 0-5°C for future use.
4. The method for preparing a nanoprobe for rapid detection of procalcitonin according to claim 3, characterized in that: Step S3 is specifically as follows: S3. Synthesize donor probe B-CDs-Ab1: S3-1, take 0.05-0.2g of donor fluorescent nanoparticles B-CDs and dissolve it in 5-20mL of 0.01M PBS solution, filter it using a 0.2-0.3μm microporous membrane, discard the filter residue, and obtain a B-CDs solution; S3-2, add 10-40 mg EDC and 5-20 mg NHS to 0.5-2 mL B-CDs solution, shake and incubate for 15-60 min at room temperature on a shaker, then add 12.5-50 μL antibody 1 solution to the B-CDs solution, and incubate for 2-8 hours on a shaker at room temperature; The antibody 1 solution is an Ab1 solution with a concentration of 0.25-1 mg / mL prepared with 0.01 M PBS; S3-3. Take 150-600 μL of the product obtained in step S3-2 and add it to an ultrafiltration centrifuge tube. Centrifuge at 5000-20000 rpm for 30-90 min. Keep the supernatant to obtain B-CDs-Ab1 concentrate. Add 150-600 μL of PBS buffer solution and centrifuge again to remove EDC, NHS and antibody 1 that have not participated in the reaction to obtain the donor probe: B-CDs-Ab1. Store at 0-5°C for future use.
5. The method for preparing a nanoprobe for rapid detection of procalcitonin according to claim 4, characterized in that: Step S4 is specifically as follows: S4. Synthetic receptor probe G-CDs-Ab2: S4-1, taking 0.05-0.2g of acceptor fluorescent nanoparticles G-CDs and dissolving them in 5-20mL 0.01M PBS solution, filtering them through a 0.2-0.3μm microporous membrane, discarding the filter residue, and obtaining a G-CDs solution; S4-2, add 10-40 mg EDC and 5-20 mg NHS to 0.5-2 mL G-CDs solution, shake and incubate for 15-60 min at room temperature on a shaker, then add 12.5-50 μL antibody 2 solution to the G-CDs solution, and incubate at 0-5°C on a shaker for 10-40 hours; The antibody 2 solution is an Ab2 solution with a concentration of 0.25-1 mg / mL prepared with 0.01 M PBS; S4-3. Take 150-600 μL of the product obtained in step S4-2 and add it to an ultrafiltration centrifuge tube. Centrifuge at 5000-20000 rpm for 30-90 min. Keep the supernatant to obtain B-CDs-Ab1 concentrate. Add 150-600 μL of PBS buffer solution and centrifuge again to remove EDC, NHS and antibody 2 that have not participated in the reaction to obtain the receptor probe: G-CDs-Ab2. Store at 0-5°C for later use.
6. The method for preparing a nanoprobe for rapid detection of procalcitonin according to claim 1, characterized in that: The following steps are involved: S1. Synthesis of donor fluorescent nanoparticles B-CDs: S1-1, dissolve 1.051 g of citric acid powder in 15 mL of purified water under magnetic stirring until the solution becomes transparent; then, add 335 μL of ethylenediamine to form a uniform mixed solution 1; S1-2, adding the mixed solution 1 into a high-pressure reactor, reacting at 180° C. for 6 h, cooling to room temperature after the reaction, filtering the obtained product solution using a 0.22 μm microporous membrane, mixing the obtained filtrate with 2 g of silica gel, and evaporating using a rotary evaporator to obtain a crude product powder 1; S1-3. Purify the crude product powder 1 by a chromatographic column separation method, use a mixture of methanol and dichloromethane in a volume ratio of 1:10 as an eluent for gradient elution, mix the collected elution solution with 2g of silica gel and perform a second spin evaporation, and finally obtain donor fluorescent nanoparticles: B-CDs, which are stored at 4°C for future use; S2. Synthesis of acceptor fluorescent nanoparticles G-CDs: Dissolve 0.5 g of passionflower extract powder and 0.8 g of m-phenylenediamine powder in 30 mL of purified water under magnetic stirring to obtain a mixed solution 2; The mixed solution 2 was added to a high-pressure reactor and reacted at 160° C. for 8 h. After the reaction was completed, the mixture was cooled to room temperature. The obtained product solution was centrifuged at 10,000 rpm for 30 min. The supernatant obtained by centrifugation was mixed with 2 g of silica gel and then evaporated using a rotary evaporator to obtain a crude product powder 2. The crude product powder 1 was purified by chromatographic column separation method, and a mixture of methanol and dichloromethane in a volume ratio of 1:10 was used as an eluent for gradient elution. The collected elution solution was mixed with 2g of silica gel for a second spin evaporation, and finally the receptor fluorescent nanoparticles: G-CDs were obtained, which were stored at 4°C for future use; S3. Synthesize donor probe B-CDs-Ab1: S3-1, take 0.1g of donor fluorescent nanoparticles B-CDs and dissolve it in 10mL of 0.01M PBS solution, filter it using a 0.22μm microporous membrane, discard the filter residue, and obtain a B-CDs solution; S3-2, add 20 mg EDC and 10 mg NHS to 1 mL B-CDs solution, shake and incubate for 30 min at room temperature on a shaker, then add 25 μL antibody 1 solution to the B-CDs solution, and incubate for 4 h at room temperature on a shaker; The antibody 1 solution is a 0.5 mg / mL Ab1 solution prepared with 0.01 M PBS; S3-3. Take 300 μL of the product obtained in step S3-2 and add it to an ultrafiltration centrifuge tube. Centrifuge at 10,000 rpm for 45 min. Keep the supernatant to obtain B-CDs-Ab1 concentrate. Add 300 μL of PBS buffer solution and centrifuge again to remove EDC, NHS and antibody 1 that have not participated in the reaction to obtain the donor probe: B-CDs-Ab1. Store at 4°C for future use. S4. Synthetic receptor probe G-CDs-Ab2: S4-1, take 0.1g of acceptor fluorescent nanoparticles G-CDs and dissolve it in 10mL of 0.01M PBS solution, filter it with a 0.22μm microporous membrane, discard the filter residue, and obtain a G-CDs solution; S4-2, add 20 mg EDC and 10 mg NHS to 1 mL G-CDs solution, shake and incubate for 30 min at room temperature on a shaker, then add 25 μL antibody 2 solution to the G-CDs solution and incubate for 20 h on a shaker at 4 °C; The antibody 2 solution is an Ab2 solution prepared with 0.01M PBS at a concentration of 0.5 mg / mL; S4-3. Take 300 μL of the product obtained in step S4-2 and add it to an ultrafiltration centrifuge tube. Centrifuge at 10,000 rpm for 45 min. Keep the supernatant to obtain B-CDs-Ab1 concentrate. Add 300 μL of PBS buffer solution and centrifuge again to remove EDC, NHS and antibody 2 that have not participated in the reaction to obtain the receptor probe: G-CDs-Ab2. Store at 4°C for future use.
7. A nanoprobe for rapid detection of procalcitonin, characterized in that: It is prepared by the method according to any one of claims 1 to 6.
8. A method for rapid detection of procalcitonin, the method using the nanoprobe as claimed in claim 7 for detection, the method comprising: The donor probe and the acceptor probe are mixed with the test solution containing PCT antigen, incubated, and the fluorescence intensity of the emission light of the reaction product under the excitation light at 450nm and 505nm is detected, which are recorded as F 450 and F 505 , calculate F 505 / F 450 The value of F was then calculated based on the pre-established relationship between PCT antigen concentration and F 505 / F 450 The PCT antigen concentration in the test solution is calculated based on the standard curve of the relationship between the values of 9. The method for rapid detection of procalcitonin according to claim 8, characterized in that: The method is: The donor probe, the acceptor probe and the test solution containing PCT antigen were mixed and incubated at 37°C for 30 min. The fluorescence intensity of the reaction product under the excitation light of 464 nm, the emission light at 450 nm and 505 nm were detected, which were recorded as F 450 and F 505 , calculate F 505 / F 450 The value of F was then calculated based on the pre-established relationship between PCT antigen concentration and F 505 / F 450 The PCT antigen concentration in the test solution is calculated based on the standard curve of the relationship between the values of 10. The method for rapid detection of procalcitonin according to claim 8, characterized in that: in, The method for constructing a standard curve includes the following steps: 1) using antigen-antibody diluent and PCT antigen to prepare a series of working solutions containing different PCT antigen concentrations; 2) adding the donor probe and the acceptor probe to the antigen-antibody dilution solution to obtain a probe solution, and preparing several equal volumes of the probe solution; 3) Add the same volume of working solution with different concentrations to each probe solution, incubate the resulting mixed solution, and detect the fluorescence intensity of the emission light of the reaction product under the excitation light at 450nm and 505nm, which are recorded as F 450 and F 505 , with PCT antigen concentration as the horizontal axis, F 505 / F 450 The value is the ordinate, and the fitting results are used to characterize the relationship between PCT antigen concentration and F 505 / F 450 The standard curve of the relationship between the values of The antigen-antibody diluent is a PBS solution containing BSA.
11. The method for rapid detection of procalcitonin according to claim 10, characterized in that: in, The method for constructing a standard curve includes the following steps: 1) Using antigen-antibody diluent and PCT antigen, prepare working solutions with PCT antigen concentrations of 0, 0.1, 0.5, 1, 5, 10, 20, 40, 60, and 80 ng / mL, respectively; 2) Add 20 μL of donor probe and 20 μL of acceptor probe to 180 μL of antigen-antibody diluent to obtain a probe solution, and prepare several aliquots of the probe solution; 3) 100 μL of working solution of different concentrations was added to each probe solution, and the resulting mixed solution was incubated at 37°C for 30 min. The fluorescence intensity of the reaction product under the excitation light of 464 nm, the emission light at 450 nm and 505 nm was detected, which were recorded as F 450 and F 505 , with PCT antigen concentration as the horizontal axis, F 505 / F 450 The value is the ordinate, and the fitting results are used to characterize the relationship between PCT antigen concentration and F 505 / F 450 The standard curve of the relationship between the values of The antigen-antibody diluent is a PBS solution containing 3 wt % BSA at a concentration of 0.01 M.