CN114106813B - Fluorescent detection of 2019-nCoV mAb composition and its preparation method and application - Google Patents

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

Fluorescent detection of 2019-nCoV mAb composition and its preparation method and application

technical field

The present 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 and its preparation method and application.

Background technique

2019-nCoV pneumonia is a virus dominated by lung disease, which may cause damage to the digestive system and nervous system, and may even lead to the death of patients. RT-PCR nucleic acid detection technology has high sensitivity and high specificity, and can detect infection earlier. However, the results of RT-PCR nucleic acid detection are affected by many factors and multiple links, such as the accuracy and reproducibility of the kit, the type of specimen, the storage and transportation of RNA is easy to degrade, the patient's infection cycle and personnel operation wait. This situation increases the risk of false negatives. The normal immune system will produce specific antibodies within 3-7 days after the invasion of the new coronavirus (2019-nCoV), and the medical diagnosis of antibody detection has been a mature clinical application, such as human immunodeficiency virus (HIV), the determination of its Antibodies are still an important basis for diagnosing AIDS.

Antibodies are tested by taking blood samples, and negative results are almost never seen. Therefore, the detection of 2019-nCoV antibody (ie 2019-nCoV mAb) can not only make up for the lack of false negative and missed detection of nucleic acid detection, but also avoid the risk of aerosol infection in the throat test paper method. The study can also be the best basis for judgment in the future possible use of 2019-nCoV vaccines.

Graphene quantum dots, a new type of quantum dots, have attracted extensive research interest due to their unique properties. Compared with graphene, graphene quantum dots exhibit stronger boundary effects. In addition, graphene quantum dots also possess properties such as low cytotoxicity, good water solubility, chemical inertness, and stable photoluminescence, which make them attractive fluorescent sensing materials for bioassays.

Contents of the invention

The technical problem to be solved by the present invention is to provide a composition for rapid and efficient fluorescence detection of 2019-nCoV mAb, its preparation method and application.

In order to achieve the above purpose, according to the first aspect of the present invention, a composition for fluorescence detection of 2019-nCoV mAb is provided. The technical solution is as follows:

The composition of fluorescence detection 2019-nCoV mAb, including: a first component, the first component includes Ag@AuNPs-NCP antigen, and the Ag@Au NPs-NCP antigen has gold-silver alloy nanoparticles; the second group The second component includes graphene quantum dots.

Further, the shape of the gold-silver alloy nanoparticles is spherical, and the particle size is 20-30 nm; the shape of the graphene quantum dot is spherical, and the particle size is 15-25 nm.

Further, when the concentration of 2019-nCoV mAb is 0-10 ng/mL, the linear relationship between the reduction rate of fluorescence intensity of the composition and the logarithm of the concentration of 2019-nCoV mAb is y=0.0225x+0.1563, R ² = 0.9924.

In order to achieve the above purpose, according to the second aspect of the present invention, a preparation method of a composition for fluorescence detection of 2019-nCoV mAb is provided. The technical solution is as follows:

The preparation method of the composition of fluorescence detection 2019-nCoV mAb described in the first aspect includes preparing the first component and preparing the second component, wherein the preparation of the first component includes the steps of: (1) obtaining gold-silver alloy nano Particle solution, namely Ag@Au NPs solution; (2) Mix Ag@Au NPs solution with NCP antigen solution, and stir at low temperature to obtain a mixed solution; (3) Add BSA solution to the mixed solution, continue to stir and centrifuge to obtain a precipitate ; (4) Disperse the precipitate in PBS solution to obtain Ag@Au NPs-NCP antigen.

Further, steps (2)-(4) were carried out at 4°C and stirred for 3-5 hours; the pH of the PBS solution was 7.5; the Ag@AuNPs-NCP antigen was stored at 4°C.

Further, the preparation of Ag@Au NPs solution includes steps:

(1) configure HAuCl ₄ solution and be heated to boiling;

(2) Add AgNO _solution in boiling liquid;

(3) Continue to add sodium citrate solution, and the Ag@Au NPs solution is obtained after the reaction is completed.

Further, preparing the second component comprises the steps of:

(1) heating citric acid to form orange liquid;

(2) Add the orange liquid into the lye and adjust the pH to obtain a GQDs solution containing graphene quantum dots.

Further, the GQDs solution was stored at 4°C; the pH was 8.

In order to achieve the above purpose, according to a third aspect of the present invention, a method for using a composition for fluorescence detection of 2019-nCoV mAb is provided. The technical solution is as follows:

The use method of the composition of the fluorescent detection 2019-nCoV mAb described in the first aspect comprises steps: (1) incubate after mixing the first component with the 2019-nCoV mAb solution; The second component is added to the solution, followed by fluorescence detection.

Further, the incubation time is ≥2.5h.

It can be seen that the preparation method of the composition of the fluorescence detection 2019-nCoV mAb of the present invention has a simple process, easy access to raw materials, low cost, and the prepared composition is easy to use, and has high selectivity and good sensitivity when detecting 2019-nCoV mAb. accuracy.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. 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.

Description of drawings

The accompanying drawings constituting a part of the present invention are used to assist the understanding of the present invention, and the content provided in the accompanying drawings and related descriptions in the present invention can be used to explain the present invention, but do not constitute an improper limitation to the present invention. In the attached picture:

Fig. 1 is a distribution diagram of ΔF/F ₀ of GQDs when the composition obtained when the pH of the PBS solution is different is used.

Fig. 2 is a distribution diagram of ΔF/F ₀ of GQDs when the obtained composition is used at different incubation times.

Figure 3 is the ultraviolet absorption spectrum of GQDs solution (a), Ag@Au NPs solution (b), GQDs solution and Ag@Au NPs solution (c), the line (d) is composed of line (a) and line ( b) Obtained by superposition.

Figure 4 shows the fluorescence emission spectrum (a) of the GQDs solution and the UV absorption spectrum (b) of the Ag@Au NPs solution.

Figure 5 is a TEM photo of GQDs powder.

Figure 6 is the TEM photo of Ag@Au NPs powder.

Fig. 7 is a spectrum diagram of Ag3d of XPS of gold-silver alloy nanoparticle powder.

Fig. 8 is the XPS spectrum of Au4f of the gold-silver alloy nanoparticle powder.

Figure 9 shows GQDs solution (a), the first mixed solution (b) composed of GQDs solution and Ag@Au NPs solution, the second mixed solution (c) composed of GQDs solution and Ag@Au NPs-NCP solution, and the second mixed solution The continuous fluorescence spectrum of the GQDs of the third mixture (d) composed of solution and 2019-nCoV mAb solution.

Figure 10 is a continuous fluorescence spectrum of GQDs after reacting different concentrations of 2019-nCoV mAb solution and composition under incubation conditions.

FIG. 11 is a linear calibration diagram obtained from FIG. 10 .

Fig. 12 is a distribution diagram of ΔF/F ₀ of GQDs after the composition reacts with different test substance solutions.

Detailed ways

The present invention will be clearly and completely described below in conjunction with the accompanying drawings. Those skilled in the art will be able to implement the present invention based on these descriptions. Before the present invention is described in conjunction with the accompanying drawings, it should be pointed out that:

The technical solutions and technical features provided in each part of the present invention, including the following description, can be combined with each other under the condition of no conflict.

In addition, the embodiments of the present invention referred to in the following description are generally only some embodiments of the present invention, not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

About terms and units in the present invention. The terms "comprising", "having" and any variations thereof in the description and claims of the present invention and related parts are intended to cover a non-exclusive inclusion.

The specific embodiment of the preparation method of the composition of the fluorescence detection 2019-nCoV mAb of the present invention includes preparing the first component and preparing the second component;

Preparation of the first component includes the steps of:

(1) Obtain a gold-silver alloy nanoparticle solution, i.e. Ag@Au NPs solution; preferably but not limited to include the following steps:

(1.1) Add 0.309mL, 30mmol/L HAuCl ₄ (auric acid chloride) aqueous solution to 50mL water, then heat to boiling;

(1.2) Add 0.198mL, 20mmol/L AgNO ₃ (silver nitrate) aqueous solution to the boiling liquid;

(1.3) Add 2.5 mL of sodium citrate solution with a mass fraction of 1% and reflux for 30 minutes. After the reaction is completed, an Ag@Au NPs solution containing gold-silver alloy nanoparticles (hereinafter referred to as Ag@Au NPs) is obtained; Ag @Au NPs directly participate in subsequent reactions in the state of solution, which can significantly improve the uniformity of the reaction; Ag@Au NPs powder can be obtained by solid-liquid separation of Ag@Au NPs solution.

(2) Mix 3 mL of Ag@Au NPs solution with 300 μL, 5 μg/mL NCP antigen solution, and stir at low temperature to obtain a mixed solution;

(3) Add 20 μL of BSA (bovine serum albumin) solution with a mass fraction of 1% to the mixture, continue to stir and centrifuge to obtain a precipitate;

(4) Disperse the precipitate in 1.5mL, 0.01mol/L PBS solution (phosphate buffered saline solution) to obtain Ag@Au NPs-NCP antigen (hereinafter directly referred to as Ag@Au NPs-NCP). Ag@Au NPs-NCP solution.

Among them, steps (2)-(4) were carried out at 4°C to ensure the activity of 2019-nCoV mAb; similarly, the prepared Ag@Au NPs-NCP solution was stored at 4°C and used whenever it was taken.

The stirring time of step (2) and step (3) is preferably 3-5 hours to make the reaction sufficient.

Preparation of the second component includes the steps of:

(1) Heat 2 g of citric acid at 200° C. for 30 minutes, so that the colorless citric acid solid gradually turns into an orange liquid;

(2) Add the orange liquid to 100 mL, 10 mg/mL NaOH solution, adjust the pH to 8, and obtain a GQDs solution containing graphene quantum dots (hereinafter referred to as GQDs). GQDs directly participate in the follow-up reaction in the state of solution, which can significantly improve the uniformity of the reaction; GQDs powder can be obtained by solid-liquid separation of GQDs solution.

The resulting GQDs solution was stored at 4°C to avoid destroying the activity of 2019-nCoV mAb when used.

Thus, the composition of the fluorescence detection 2019-nCoV mAb of the present invention is obtained, and the composition includes a first component and a second component; the first component is Ag@Au NPs-NCP solution; the second component is Ag@Au NPs-NCP solution; The two components are GQDs solution.

The method for using the composition of the fluorescent detection 2019-nCoV mAb comprises steps:

(1) Mix 100 μL of Ag@Au NPs-NCP solution with 20 μL of 2019-nCoV mAb solution and incubate;

(2) After the incubation was completed, 20 μL of GQDs solution was added to the incubation, and then fluorescence detection was performed.

The beneficial effects of the present invention are illustrated below by examples.

First, seven kinds of Ag@Au NPs-NCP solutions were prepared under the conditions of PBS solution with pH of 5.5, 6, 6.5, 7, 7.5, 8 and 8.5, respectively. First, the fluorescence intensity of the pure GQDs solution was tested ( F ₀ ), then the fluorescence intensity (F ₁ ) of the GQDs after the reaction of the composition with the 2019-nCoV mAb at a concentration of 10 ng/mL was tested.

Fig. 1 is a distribution diagram of ΔF/F ₀ of GQDs when the composition obtained when the pH of the PBS solution is different is used. Wherein, ΔF=F ₀ -F ₁ .

It can be seen from FIG. 1 that when the pH of the PBS solution is 7.5, the obtained composition can obtain the best detection effect.

Secondly, seven kinds of Ag@Au NPs-NCP solutions were prepared under the condition of incubation time of 0.5h, 1h, 1.5h, 2h, 2.5h, 3h and 3.5h, respectively, and the corresponding F ₁ were tested.

Fig. 2 is a distribution diagram of ΔF/F ₀ of GQDs when the obtained composition is used at different incubation times.

It can be seen from Figure 2 that when the incubation time is ≥ 2.5h, the obtained composition can achieve better detection results, and in terms of efficiency, the best incubation time is 2.5h.

The characterization results and performance test results of GQDs solution, Ag@Au NPs solution and Ag@Au NPs-NCP solution prepared by optimal process parameters are further described below.

Figure 3 is the ultraviolet absorption spectrum of GQDs solution (a), Ag@Au NPs solution (b), GQDs solution and Ag@Au NPs solution (c), the line (d) is composed of line (a) and line ( b) Obtained by superposition. Figure 4 shows the fluorescence emission spectrum (a) of the GQDs solution and the UV absorption spectrum (b) of the Ag@Au NPs solution.

Figure 3(a) has an obvious π-π* absorption peak at 365 nm, which is related to the C=C transition of GQDs. It can be seen from Figure 4(a) that when the excitation light wavelength is 390nm, the GQDs solution has a maximum emission spectrum at 465nm; Figure 3(a) and Figure 4(a) show that GQDs have been successfully synthesized.

From Figures 3(b) and 4(b), it can be seen that the Ag@Au NPs solution has an obvious UV absorption peak near 490 nm, indicating that Ag@Au NPs have been successfully synthesized.

It can also be seen from Figure 4 that the absorbance of line (d) at the same wavelength is significantly greater than that of line (c), indicating that there is an interaction between GQDs and Ag@Au NPs; line (a) and line (b) partially overlap, It shows that there is fluorescence resonance energy transfer between GQDs and Ag@Au NPs.

Figure 5 is a TEM photo of GQDs powder. Figure 6 is the TEM photo of Ag@Au NPs powder.

It can be seen from Figure 5 that the morphology of GQDs is spherical with a particle size of 15-25 nm and good dispersion; it can be seen from Figure 6 that the morphology of Ag@Au NPs is spherical with a particle size of 20-30 nm.

Fig. 7 is a spectrum diagram of Ag3d of XPS of gold-silver alloy nanoparticle powder. Fig. 8 is the XPS spectrum of Au4f of the gold-silver alloy nanoparticle powder.

It can be seen from Fig. 7 that the characteristic peaks at binding energies of 367.7 eV and 373.7 eV correspond to Ag3d _3/2 and Ag3d _5/2 , respectively, which are attributed to the metallic silver in Ag@Au NPs. It can be seen from Fig. 8 that the characteristic peaks at binding energies of 83.6 eV and 87.3 eV correspond to Au4f _7/2 and Au4f _5/2 , respectively, which are attributed to the metallic gold in Ag@Au NPs.

Figure 9 shows GQDs solution (a), the first mixed solution (b) composed of GQDs solution and Ag@Au NPs solution, the second mixed solution (c) composed of GQDs solution and Ag@Au NPs-NCP solution, and the second mixed solution The continuous fluorescence spectrum of the GQDs of the third mixture (d) composed of solution and 2019-nCoV mAb solution.

It can be seen from Figure 9 that at the same wavelength, the fluorescence intensity of pure GQDs is the highest; in line (c), when Ag@Au NPs-NCP solution is added, the fluorescence intensity of GQDs decreases significantly; but compared with line (c) It can be seen from line (d) that when 2019-nCoV mAb was added, the fluorescence intensity of GQDs recovered significantly.

The reason for the recovery of the fluorescence intensity of GQDs is: the fluorescence intensity of pure GQDs is higher, but when GQDs react with Ag@AuNPs, the fluorescence intensity of GQDs will decrease due to the fluorescence resonance energy transfer effect; when Ag@Au NPs and NCP After antigen binding, the distance between GQDs and Ag@Au NPs-NCP increases until the optimal distance for fluorescence resonance energy transfer is reached, at which point the FRET efficiency reaches the maximum and the fluorescence intensity of GQDs decreases further; however, when 2019- After nCoV mAb, due to the steric hindrance effect of 2019-nCoV mAb, the fluorescence of GQDs recovered to a certain extent. It can be seen that the amount of 2019-nCoV mAb added can be judged by the degree of recovery of the fluorescence intensity of GQDs before and after the addition of 2019-nCoV mAb.

In view of the significant impact of the above incubation conditions on the fluorescence intensity of GQDs, the incubation method was used to test the linear relationship between the composition and the concentration of the test substance. Obviously, when the GQDs solution reacts with the mixture of the incubated Ag@Au NPs-NCP solution and the detection substance, the detection substance in the mixture can also increase the distance between the GQDs and the Ag@Au NPs-NCP The steric hindrance effect is generated, which leads to the reduction of the fluorescence of GQDs to varying degrees; if the concentration of the detection substance 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 , when the composition is used under incubation conditions, the concentration of the detection substance can be judged by the decrease rate of the fluorescence intensity of the GQDs.

Figure 10 is a continuous fluorescence spectrum of GQDs after reacting different concentrations of 2019-nCoV mAb solution and composition under incubation conditions. FIG. 11 is a linear calibration diagram obtained from FIG. 10 . Among them, the concentration of 2019-nCoV mAb solution is 0, 1×10 ^-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 Figure 10, as the concentration of 2019-nCoV mAb increased, the fluorescence intensity of the reacted GQDs gradually increased, which indicated that the effect of Ag@Au NPs-NCP solution on GQDs changed with the increase of 2019-nCoV mAb concentration. Weak, so that the reduction rate of the fluorescence intensity of GQDs is reduced.

After processing Figure 10, it can be concluded that when the concentration of the 2019-nCoV mAb solution is 0-10 ng/mL, the reduction rate of the fluorescence intensity of the GQDs when the composition is used (in the y-axis, represented by ΔF/F ₀ ) The linear relationship with the logarithm of the concentration of 2019-nCoV mAb (on the x-axis, represented by logC _{(2019-nCoVmAb)} ) is y=0.0225x+0.1563, R ² =0.9924, and the detection limit is 50 fg mL ^-1 .

Fig. 12 is a distribution diagram of ΔF/F ₀ of GQDs after the composition reacts with different test substance solutions.

As can be seen from Figure 12, compared with bovine serum albumin, glutathione, L-cysteine, glucose, L-histidine and folic acid, the composition of the present invention has an excellent effect on 2019-nCoV mAb. selectivity.

The content related to the present invention has been described above. Those skilled in the art will be able to implement the present invention based on these descriptions. Based on the above content of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

Claims (8)

1. The composition of fluorescence detection 2019-nCoV mAb is characterized in that: comprising: a first component, the first component includes Ag@Au NPs-NCP antigen, and the Ag@Au NPs-NCP antigen has gold-silver alloy nanoparticles; a second component, the second component comprising graphene quantum dots; The preparation method of the composition of fluorescence detection 2019-nCoV mAb comprises the preparation of the first component and the preparation of the second component, wherein the preparation of the first component comprises the steps of: (1) Obtain a gold-silver alloy nanoparticle solution, namely Ag@Au NPs solution; (2) Mix the Ag@Au NPs solution with the NCP antigen solution, and stir at low temperature to obtain a mixed solution; (3) Add BSA solution to the mixture, continue to stir and centrifuge to obtain the precipitate; (4) Disperse the precipitate in PBS solution to obtain Ag@Au NPs-NCP antigen; The preparation of Ag@Au NPs solution includes steps: (1) Prepare HAuCl ₄ solution and heat to boiling; (2) Add AgNO ₃ solution to the boiling liquid; (3) Continue to add sodium citrate solution, and the Ag@Au NPs solution is obtained after the reaction is completed; Preparation of the second component includes the steps of: (1) Heat citric acid to form an orange liquid; (2) Add the orange liquid to the lye and adjust the pH to obtain a GQDs solution containing graphene quantum dots. 2. The composition of fluorescence detection 2019-nCoV mAb as claimed in claim 1, characterized in that: the appearance of gold-silver alloy nanoparticles is spherical, and the particle size is 20-30nm; the appearance of graphene quantum dots is spherical, The particle size is 15-25nm. 3. The composition for fluorescence detection of 2019-nCoV mAb according to claim 1, characterized in that: when the concentration of 2019-nCoV mAb is 0-10 ng/mL, the decrease rate of fluorescence intensity of the composition is the same as that of 2019-nCoV mAb The linear relationship of the logarithm of the concentration is y=0.0225x+0.1563, R ² =0.9924. 4. The method for preparing the composition of fluorescence detection 2019-nCoV mAb according to any one of claims 1 to 3, comprising preparing the first component and preparing the second component, wherein, Preparation of the first component includes the steps of: (1) Obtain a gold-silver alloy nanoparticle solution, namely Ag@Au NPs solution; (2) Mix the Ag@Au NPs solution with the NCP antigen solution, and stir at low temperature to obtain a mixed solution; (3) Add BSA solution to the mixture, continue to stir and centrifuge to obtain the precipitate; (4) Disperse the precipitate in PBS solution to obtain Ag@Au NPs-NCP antigen; The preparation of Ag@Au NPs solution includes steps: (1) Prepare HAuCl ₄ solution and heat to boiling; (2) Add AgNO ₃ solution to the boiling liquid; (3) Continue to add sodium citrate solution, and the Ag@Au NPs solution is obtained after the reaction is completed; Preparation of the second component includes the steps of: (1) Heat citric acid to form an orange liquid; (2) Add the orange liquid to the lye and adjust the pH to obtain a GQDs solution containing graphene quantum dots. 5. The preparation method of the composition of fluorescence detection 2019-nCoV mAb according to claim 4, characterized in that: the steps (2)-(4) of preparing the first component are carried out at 4°C and stirred for 3-5 hours; The pH of the PBS solution was 7.5; the Ag@Au NPs-NCP antigen was stored at 4°C. 6. The preparation method of the composition for fluorescence detection of 2019-nCoV mAb according to claim 4, characterized in that: the GQDs solution is stored at 4°C; the pH is 8. 7. The method for using the composition of the fluorescent detection 2019-nCoV mAb according to any one of claims 1 to 3, comprising the steps of: (1) Mix the first component with the 2019-nCoV mAb solution and incubate; (2) After the incubation is completed, add the second component to the incubation, and then perform fluorescence detection. 8. The method of using the composition for fluorescence detection of 2019-nCoV mAb according to claim 7, characterized in that: incubation time ≥ 2.5h.

Images