CN114106813B - Composition for fluorescence detection of 2019-nCoV mAb, preparation method and application thereof - Google Patents
Composition for fluorescence detection of 2019-nCoV mAb, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a composition for fluorescence detection of 2019-nCoV mAb, a preparation method and application thereof. The composition comprises: a first component comprising ag@au NPs-NCP antigen having gold-silver alloy nanoparticles; and a second component comprising graphene quantum dots. The method of preparing the composition comprises preparing a first component and preparing a second component, wherein the preparing the first component comprises the steps of: (1) Obtaining gold-silver alloy nanoparticle solution, namely Ag@Au NPs solution; (2) Mixing Ag@Au NPs solution with NCP antigen solution, and stirring at low temperature to obtain mixed solution; (3) Adding BSA solution into the mixed solution, continuously stirring, and centrifugally separating to obtain a precipitate; (4) Dispersing the precipitate in PBS solution to obtain Ag@Au NPs-NCP antigen. Therefore, the preparation method of the composition for fluorescence detection of 2019-nCoV mAb has the advantages of simple process, easily obtained raw materials, low cost, convenient use of the prepared composition, high selectivity and good accuracy in detection of 2019-nCoV mAb.
Description
Technical Field
The invention relates to the technical field of 2019 novel coronavirus antibody (2019-nCoV mAb) detection, in particular to a composition for fluorescence detection of 2019-nCoV mAb, a preparation method and application thereof.
Background
The novel coronavirus pneumonia is a virus mainly used for pulmonary diseases, and can cause damage to digestive systems and nervous systems and even cause death of patients. The RT-PCR nucleic acid detection technology has high sensitivity and high specificity, and can discover infection earlier. However, the results of RT-PCR nucleic acid detection are affected by a variety of factors and links, such as the accuracy and reproducibility of the kit, the type of specimen, the ease of storage and transport of RNA for degradation, the period of infection and personnel handling in patients, etc. This situation increases the risk of false negatives. The normal immune system produces specific antibodies within 3-7 days after the invasion of the novel coronavirus (2019-nCoV), and medical diagnosis of antibody detection is already a mature clinical application, such as Human Immunodeficiency Virus (HIV), and the determination of antibodies is still an important basis for diagnosing aids.
The antibody is detected by the blood sampling sample, and a negative result hardly appears. Therefore, the detection of 2019-nCoV antibody (namely 2019-nCoV mAb) can compensate the missing of false negative detection in nucleic acid detection, and simultaneously avoid the risk of aerosol infection in the detection of pharynx test paper method accounting. The study also provides the best basis for future judgment in the possible use of 2019-nCoV vaccine.
Graphene quantum dots are a novel quantum dot, and have attracted extensive research interest due to their unique properties. Compared with graphene, the graphene quantum dots exhibit stronger boundary effects. In addition, graphene quantum dots have the characteristics of low cytotoxicity, good water solubility, chemical inertness, stable photoluminescence and the like, and the characteristics make the graphene quantum dots attractive fluorescent sensing materials for bioassays.
Disclosure of Invention
The invention aims to provide a rapid and efficient composition for detecting 2019-nCoV mAb by fluorescence, a preparation method and application thereof.
To achieve the above object, according to a first aspect of the present invention, there is provided a composition for fluorescence detection of 2019-nCoV mAb. The technical proposal is as follows:
a composition for fluorescence detection of 2019-nCoV mAb comprising: a first component comprising ag@au NPs-NCP antigen having gold-silver alloy nanoparticles; and a second component comprising graphene quantum dots.
Further, the gold-silver alloy nano particles are spherical in shape and have the granularity of 20-30 nm; the shape of the graphene quantum dot is spherical, and the granularity is 15-25 nm.
Further, when the concentration of 2019-nCoV mAb is 0 to 10ng/mL, the linear relationship between the fluorescence intensity decrease rate of the composition and the logarithm of the concentration of 2019-nCoV mAb is y=0.0225x+0.1563, R 2 ＝0.9924。
To achieve the above object, according to a second aspect of the present invention, there is provided a method for preparing a composition for fluorescence detection of 2019-nCoV mAb. The technical proposal is as follows:
the method of preparing a composition for fluorescence detection of 2019-nCoV mAb of the first aspect comprising preparing a first component and preparing a second component, wherein preparing the first component comprises the steps of: (1) Obtaining gold-silver alloy nanoparticle solution, namely Ag@Au NPs solution; (2) Mixing Ag@Au NPs solution with NCP antigen solution, and stirring at low temperature to obtain mixed solution; (3) Adding BSA solution into the mixed solution, continuously stirring, and centrifugally separating to obtain a precipitate; (4) Dispersing the precipitate in PBS solution to obtain Ag@Au NPs-NCP antigen.
Further, the steps (2) - (4) are carried out at the temperature of 4 ℃ and stirred for 3-5 h; the pH of the PBS solution was 7.5; ag@Au NPs-NCP antigen was stored at 4 ℃.
Further, the preparation of the Ag@Au NPs solution comprises the following steps:
(1) Configuration of HAuCl 4 The solution is heated to boiling;
(2) AgNO is added into the boiling liquid 3 A solution;
(3) Continuously adding sodium citrate solution, and obtaining Ag@Au NPs solution after the reaction is completed.
Further, the preparation of the second component comprises the steps of:
(1) Heating citric acid to form an orange liquid;
(2) And adding the orange liquid into alkali liquor, and regulating the pH value to obtain the GQDs solution containing the graphene quantum dots.
Further, the GQDs solution is preserved at 4 ℃; the pH was 8.
To achieve the above object, according to a third aspect of the present invention, there is provided a method of using a composition for fluorescence detection of 2019-nCoV mAb. The technical proposal is as follows:
the method of using the composition for fluorescence detection of 2019-nCoV mAb of the first aspect comprising the steps of: (1) Incubating the first component after mixing with 2019-nCoV mAb solution; (2) After the incubation is completed, a second component is added to the incubation and then fluorescence detection is performed.
Further, the incubation time is more than or equal to 2.5 hours.
Therefore, the preparation method of the composition for fluorescence detection of 2019-nCoV mAb has the advantages of simple process, easily obtained raw materials, low cost, convenient use of the prepared composition, high selectivity and good accuracy in detection of 2019-nCoV mAb.
The invention is further described below with reference to the drawings and detailed description. Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The accompanying drawings, which form a part hereof, are shown by way of illustration and not of limitation, and in which are shown by way of illustration and description of the invention. In the drawings:
FIG. 1 shows the ΔF/F of GQDs when the resulting composition is used with different pH of PBS solution 0 Is a profile of (a).
FIG. 2 shows the ΔF/F of GQDs when the resulting composition is used with different incubation times 0 Is a profile of (a).
FIG. 3 shows the ultraviolet absorption spectra of a mixture (c) of GQDs solution (a), ag@Au NPs solution (b), GQDs solution and Ag@Au NPs solution, and line (d) is obtained by superimposing line (a) and line (b).
FIG. 4 shows fluorescence emission spectra (a) of GQDs solutions and ultraviolet absorption spectra (b) of Ag@Au NPs solutions.
Fig. 5 is a TEM photograph of the GQDs powder.
Fig. 6 is a TEM photograph of ag@au NPs powder.
FIG. 7 is a spectrum of XPS Ag3d for gold and silver alloy nanoparticle powder.
FIG. 8 is a spectrum of Au4f of XPS of gold-silver alloy nanoparticle powder.
FIG. 9 is a continuous fluorescence spectrum of GQDs of a first mixed solution (b) composed of GQDs solution (a), GQDs solution and Ag@Au NPs solution, a second mixed solution (c) composed of GQDs solution and Ag@Au NPs-NCP solution, and a third mixed solution (d) composed of the second mixed solution and 2019-nCoV mAb solution.
FIG. 10 is a continuous fluorescence spectrum of GQDs after different concentrations of 2019-nCoV mAb solution reacted with the composition under incubation conditions.
Fig. 11 is a linear calibration plot from fig. 10.
FIG. 12 shows the ΔF/F of GQDs after reaction of the composition with solutions of different analytes 0 Is a profile of (a).
Detailed Description
The present invention will now be described more fully hereinafter with reference to the accompanying drawings. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. Before describing the present invention with reference to the accompanying drawings, it should be noted in particular that:
the technical solutions and technical features provided in the sections including the following description in the present invention may be combined with each other without conflict.
In addition, the embodiments of the present invention referred to in the following description are typically only some, but not all, embodiments of the present invention. Therefore, all other embodiments, which can be made by one of ordinary skill in the art without undue burden, are intended to be within the scope of the present invention, based on the embodiments of the present invention.
Terms and units in relation to the present invention. The terms "comprising," "having," and any variations thereof in the description and claims of the invention and in the relevant sections are intended to cover a non-exclusive inclusion.
Specific embodiments of the methods of making compositions for fluorescence detection 2019-nCoV mabs of the present invention include making a first component and making a second component;
the preparation of the first component comprises the steps of:
(1) Obtaining gold-silver alloy nanoparticle solution, namely Ag@Au NPs solution; preferably, but not limited to, the steps of:
(1.1) 0.309mL of 30mmol/L HAuCl 4 The aqueous solution of (chloroauric acid) was added to 50mL of water and then heated to boiling;
(1.2) 0.198mL, 20mmol/L AgNO was added to the boiling solution 3 (silver nitrate) aqueous solution;
(1.3) adding 2.5mL of sodium citrate solution with the mass fraction of 1% and carrying out reflux reaction for 30 minutes, thus obtaining Ag@Au NPs solution containing gold-silver alloy nano particles (hereinafter referred to as Ag@Au NPs); the Ag@Au NPs directly participate in subsequent reactions in a solution state, so that the uniformity of the reactions can be remarkably improved; and carrying out solid-liquid separation on the Ag@Au NPs solution to obtain Ag@Au NPs powder.
(2) 3mL of Ag@Au NPs solution is mixed with 300 mu L of NCP antigen solution with 5 mu g/mL, and the mixture is obtained by stirring at a low temperature;
(3) Adding 20 mu L of BSA (bovine serum albumin) solution with the mass fraction of 1% into the mixed solution, continuously stirring, and centrifugally separating to obtain a precipitate;
(4) Dispersing the precipitate in 1.5mL of 0.01mol/L PBS solution (phosphate buffer solution) to obtain Ag@Au NPs-NCP solution containing Ag@Au NPs-NCP antigen (hereinafter referred to as Ag@Au NPs-NCP).
Wherein steps (2) - (4) are performed at 4 ℃ to ensure activity of 2019-nCoV mAb; similarly, the prepared Ag@Au NPs-NCP solution is stored at the temperature of 4 ℃ and is taken and used at any time.
The stirring time of step (2) and step (3) is preferably 3 to 5 hours to allow the reaction to be sufficient.
The preparation of the second component comprises the steps of:
(1) 2g of citric acid was heated at 200℃for 30 minutes to gradually change the colorless citric acid solid into an orange liquid;
(2) And adding the orange liquid into 100mL of 10mg/mL NaOH solution, and regulating the pH to 8 to obtain the GQDs solution containing the graphene quantum dots (hereinafter referred to as GQDs). GQDs directly participate in subsequent reactions in a solution state, so that the uniformity of the reactions can be remarkably improved; and carrying out solid-liquid separation on the GQDs solution to obtain GQDs powder.
The resulting GQDs solution was stored at 4deg.C to avoid disruption of 2019-nCoV mAb activity upon use.
Thus, a composition of the fluorescence detection 2019-nCoV mAb of the invention is obtained, which comprises a first component and a second component; the first component is Ag@Au NPs-NCP solution; the second component is a GQDs solution.
The method for using the composition for fluorescence detection of 2019-nCoV mAb comprises the following steps:
(1) Incubation was performed after mixing 100 μl of ag@au NPs-NCP solution with 20 μl of 2019-nCoV mAb solution;
(2) After the incubation was completed, 20. Mu.L of GQDs solution was added to the incubation, followed by fluorescence detection.
The beneficial effects of the invention are illustrated below by way of examples.
Firstly, seven Ag@Au NPs-NCP solutions were prepared at pH 5.5, 6, 6.5, 7, 7.5, 8 and 8.5, respectively, and first, the fluorescence intensity of pure GQDs solutions was tested (F 0 ) The compositions were then tested for fluorescence intensity of GQDs after reaction with 2019-nCoV mAb at a concentration of 10ng/mL (F 1 )。
FIG. 1 shows the ΔF/F of GQDs when the resulting composition is used with different pH of PBS solution 0 Is a profile of (a). Wherein Δf=f 0 -F 1 。
As can be seen from fig. 1, the resulting composition can obtain the best detection effect when the pH of the PBS solution is 7.5.
Next, seven Ag@Au NPs-NCP solutions were prepared and tested for F under conditions of incubation times of 0.5h, 1h, 1.5h, 2h, 2.5h, 3h and 3.5h, respectively 1 。
FIG. 2 shows the delta GQDs for compositions obtained at different incubation times when usedF/F 0 Is a profile of (a).
As can be seen from FIG. 2, when the incubation time is not less than 2.5 hours, the obtained composition can obtain a better detection effect, and the optimal incubation time is 2.5 hours from the aspect of efficiency.
The characterization results and performance test results of the Ag@Au NPs-NCP solution prepared by the GQDs solution, the Ag@Au NPs solution and the optimal technological parameters are further described below.
FIG. 3 shows the ultraviolet absorption spectra of a mixture (c) of GQDs solution (a), ag@Au NPs solution (b), GQDs solution and Ag@Au NPs solution, and line (d) is obtained by superimposing line (a) and line (b). FIG. 4 shows fluorescence emission spectra (a) of GQDs solutions and ultraviolet absorption spectra (b) of Ag@Au NPs solutions.
Fig. 3 (a) has a distinct pi-pi absorption peak at 365nm, which is associated with the c=c transition of GQDs. As can be seen from FIG. 4 (a), the GQDs solution has a maximum emission spectrum at 465nm when the excitation light wavelength is 390 nm; FIGS. 3 (a) and 4 (a) show that GQDs have been successfully synthesized.
As can be seen from FIGS. 3 (b) and 4 (b), the Ag@Au NPs solution has a distinct UV absorption peak around 490nm, indicating that the Ag@Au NPs have been successfully synthesized.
As can also be seen from fig. 4, the absorbance of line (d) at the same wavelength is significantly greater than the absorbance of line (c), indicating that there is an interaction between GQDs and ag@au NPs; the partial overlap of lines (a) and (b) indicates that there is fluorescence resonance energy transfer between GQDs and Ag@Au NPs.
Fig. 5 is a TEM photograph of the GQDs powder. Fig. 6 is a TEM photograph of ag@au NPs powder.
As can be seen from FIG. 5, the GQDs are spherical in shape, 15-25 nm in granularity and good in dispersibility; as can be seen from FIG. 6, the morphology of Ag@Au NPs is spherical, and the granularity is 20-30 nm.
FIG. 7 is a spectrum of XPS Ag3d for gold and silver alloy nanoparticle powder. FIG. 8 is a spectrum of Au4f of XPS of gold-silver alloy nanoparticle powder.
As seen from FIG. 7, characteristic peaks at binding energies of 367.7eV and 373.7eV, respectively, correspond to Ag3d, respectively 3/2 And Ag3d 5/2 Due to the metallic silver in ag@au NPs. From the figure8, the characteristic peaks at binding energies of 83.6eV and 87.3eV, respectively, correspond to Au4f, respectively 7/2 And Au4f 5/2 Due to the metallic gold in ag@au NPs.
FIG. 9 is a continuous fluorescence spectrum of GQDs of a first mixed solution (b) composed of GQDs solution (a), GQDs solution and Ag@Au NPs solution, a second mixed solution (c) composed of GQDs solution and Ag@Au NPs-NCP solution, and a third mixed solution (d) composed of the second mixed solution and 2019-nCoV mAb solution.
As can be seen from fig. 9, at the same wavelength, the fluorescence intensity of pure GQDs is highest; in line (c), the fluorescence intensity of GQDs is significantly reduced after the Ag@Au NPs-NCP solution is added; however, comparing line (c) with line (d) shows that there is a significant recovery in fluorescence intensity of GQDs when 2019-nCoV mAb was added.
The reason why the fluorescence intensity of the GQDs is recovered is that: the fluorescence intensity of the pure GQDs is higher, but when the GQDs react with Ag@Au NPs, the fluorescence intensity of the GQDs is reduced due to the fluorescence resonance energy transfer effect; when Ag@Au NPs are combined with NCP antigens, the distance between the GQDs and the Ag@Au NPs-NCP is increased until the optimal distance for fluorescence resonance energy transfer is reached, at the moment, the FRET efficiency reaches the maximum value, and the fluorescence intensity of the GQDs is further reduced; however, when 2019-nCoV mAb was added, fluorescence of GQDs was somewhat restored due to steric hindrance effect of 2019-nCoV mAb. From this, it can be seen that the addition amount of 2019-nCoV mAb can be judged by the degree of recovery of the fluorescence intensity of GQDs before and after the addition of 2019-nCoV mAb.
Given the above significant effect of incubation conditions on fluorescence intensity of GQDs, the compositions were tested in an incubation manner for a linear relationship with the concentration of the detector when used. Obviously, when the GQDs solution reacts with the mixed solution of the incubated Ag@Au NPs-NCP solution and the detection object, the detection object in the mixed solution can also increase the distance between the GQDs and the Ag@Au NPs-NCP to generate a steric hindrance effect, so that fluorescence of the GQDs is reduced to different degrees; if the concentration of the detection object is high, the steric hindrance effect is high, and the fluorescence intensity reduction rate of GQDs is low; otherwise, the fluorescence intensity reduction rate of GQDs is high; therefore, the concentration of the analyte can be determined by the fluorescence intensity decrease rate of the GQDs when the composition is used under the incubation condition.
FIG. 10 is a continuous fluorescence spectrum of GQDs after different concentrations of 2019-nCoV mAb solution reacted with the composition under incubation conditions. Fig. 11 is a linear calibration plot from fig. 10. Wherein the concentration of 2019-nCoV mAb solution is 0 and 1 multiplied by 10 -4 ng/mL、1×10 -3 ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, and 10ng/mL.
As shown in FIG. 10, the fluorescence intensity of the reacted GQDs gradually increased with increasing concentration of 2019-nCoV mAb, which indicates that the effect of the Ag@Au NPs-NCP solution on the GQDs was weakened with increasing concentration of 2019-nCoV mAb, thereby decreasing the fluorescence intensity decrease rate of the GQDs.
As can be seen from the treatment of FIG. 10, when the concentration of 2019-nCoV mAb solution was 0 to 10ng/mL, the fluorescence intensity decrease rate of GQDs in the composition was measured (on the y-axis, with DeltaF/F 0 Expressed) with the logarithm of the concentration of 2019-nCoV mAb (on the x-axis, using logC (2019-nCoVmAb) Expressed) is y=0.0225x+0.1563, r 2 = 0.9924, detection limit of 50fg mL -1 。
FIG. 12 shows the ΔF/F of GQDs after reaction of the composition with solutions of different analytes 0 Is a profile of (a).
As can be seen from fig. 12, the composition of the present invention has excellent selectivity for 2019-nCoV mAb compared to bovine serum albumin, glutathione, L-cysteine, glucose, L-histidine and folic acid.
The content of the present invention is described above. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. Based on the foregoing, all other embodiments that may be obtained by one of ordinary skill in the art without undue burden are within the scope of the present invention.
Claims (8)
1. A composition for fluorescence detection of 2019-nCoV mAb characterized by: comprising the following steps:
a first component comprising ag@au NPs-NCP antigen having gold-silver alloy nanoparticles;
a second component comprising graphene quantum dots;
a method of preparing a composition for fluorescence detection of 2019-nCoV mabs includes preparing a first component and preparing a second component, wherein preparing the first component includes the steps of:
(1) Obtaining gold-silver alloy nanoparticle solution, namely Ag@Au NPs solution;
(2) Mixing Ag@Au NPs solution with NCP antigen solution, and stirring at low temperature to obtain mixed solution;
(3) Adding BSA solution into the mixed solution, continuously stirring, and centrifugally separating to obtain a precipitate;
(4) Dispersing the precipitate in PBS solution to obtain Ag@Au NPs-NCP antigen;
the preparation of the Ag@Au NPs solution comprises the following steps:
(1) Configuration of HAuCl 4 The solution is heated to boiling;
(2) AgNO is added to the boiling liquid 3 A solution;
(3) Continuously adding a sodium citrate solution, and obtaining an Ag@Au NPs solution after the reaction is completed;
the preparation of the second component comprises the steps of:
(1) Heating citric acid to form an orange liquid;
(2) And adding the orange liquid into alkali liquor, and regulating the pH value to obtain the GQDs solution containing the graphene quantum dots.
2. The composition for fluorescence detection of 2019-nCoV mAb of claim 1, wherein: the gold-silver alloy nano particles are spherical in shape and have the granularity of 20-30 nm; the shape of the graphene quantum dot is spherical, and the granularity is 15-25 nm.
3. The composition for fluorescence detection of 2019-nCoV mAb of claim 1, wherein: when the concentration of 2019-nCoV mAb is 0-10 ng/mL, the linear relation between the fluorescence intensity reduction rate of the composition and the logarithm of the concentration of 2019-nCoV mAb is y=0.0225x+0.1563, R 2 =0.9924。
4. A method for preparing a composition for fluorescence detection of 2019-nCoV mAb according to any one of claim 1 to 3, comprising preparing a first component and preparing a second component, wherein,
the preparation of the first component comprises the steps of:
(1) Obtaining gold-silver alloy nanoparticle solution, namely Ag@Au NPs solution;
(2) Mixing Ag@Au NPs solution with NCP antigen solution, and stirring at low temperature to obtain mixed solution;
(3) Adding BSA solution into the mixed solution, continuously stirring, and centrifugally separating to obtain a precipitate;
(4) Dispersing the precipitate in PBS solution to obtain Ag@Au NPs-NCP antigen;
the preparation of the Ag@Au NPs solution comprises the following steps:
(1) Configuration of HAuCl 4 The solution is heated to boiling;
(2) AgNO is added to the boiling liquid 3 A solution;
(3) Continuously adding a sodium citrate solution, and obtaining an Ag@Au NPs solution after the reaction is completed;
the preparation of the second component comprises the steps of:
(1) Heating citric acid to form an orange liquid;
(2) And adding the orange liquid into alkali liquor, and regulating the pH value to obtain the GQDs solution containing the graphene quantum dots.
5. The method for preparing the composition for fluorescence detection of 2019-nCoV mAb of claim 4, which is characterized by: steps (2) - (4) for preparing the first component are carried out at 4 ℃ and stirred for 3-5 h; the pH of the PBS solution was 7.5; ag@Au NPs-NCP antigen was stored at 4 ℃.
6. The method for preparing the composition for fluorescence detection of 2019-nCoV mAb of claim 4, which is characterized by: storing GQDs solution at 4deg.C; the pH was 8.
7. A method of using the composition of fluorescence detection 2019-nCoV mAb of one of claims 1-3 comprising the steps of:
(1) Incubating the first component after mixing with 2019-nCoV mAb solution;
(2) After the incubation is completed, a second component is added to the incubation and then fluorescence detection is performed.
8. The method of using the composition for fluorescence detection of 2019-nCoV mAb of claim 7, wherein: the incubation time is more than or equal to 2.5 hours.
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