key: cord-0829909-ypplh1jg
authors: Li, Nan; Shi, Linhong; Zou, Xue; Wang, Tengfei; Wang, Dongmei; Gong, Zhengjun; Fan, Meikun
title: Fluorescence immunoassay rapid detection of 2019-nCoV antibody based on the fluorescence resonance energy transfer between graphene quantum dots and Ag@Au nanoparticle
date: 2021-12-01
journal: Microchem J
DOI: 10.1016/j.microc.2021.107046
sha: 5572358f59d6143a91ce2c1efe18dffe25728194
doc_id: 829909
cord_uid: ypplh1jg

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has afflicted the world, is a highly contagious virus. The timely and accurate diagnosis of SARS-CoV-2 infections is vital for disease control and prevention. This work is that a fluorescence immunoassay was developed to detect 2019-nCoV antibodies (2019-nCoV mAb). Fluorescent graphene quantum dots (GQDs) and Ag@Au nanoparticles (Ag@AuNPs) were successfully synthesized and characterized. Fluorescence resonance energy transfer (FRET) enables Ag@AuNPs effective quenching of GQDs fluorescence.. With the presence of 2019-nCoV mAb, a steric hindrance was observed between the Ag@AuNPs-NCP (2019-nCoV antigen) complex and GQDs, which reduced the FRET efficiency and restored the fluorescence of GQDs. The fluorescence enhancement efficiency has a satisfactory linear relationship with the logarithm of the 2019-nCoV mAb in a concentration scope of 0.1 pg mL(-1) - 10 ng mL(-1), and the limit of detection was 50 fg mL(-1). This study tested other proteins and proved that the fluorescence immunoassay has good selectivity. When this method was applied to the standard addition recovery trial test of 2019-nCoV mAb in serum samples, the recovery rate was between 90.8% and 103.3%, and satisfactory results were obtained.

Coronavirus disease 2019 (COVID- 19) , Novel coronavirus pneumonia is an acute infectious disease caused by SARS-CoV-2. A new infectious disease, 2019-nCoV, which is dominated by lung diseases, has a terrible rate of transmission, and can cause damage to the digestive system and nervous system. The disease can lead to the death of the patient [1] . Therefore, the effective detection of 2019-nCoV is very important. Nucleic acid testing techniques for Reverse Transcription-Polymerase Chain Reaction (RT-PICR) are clinical testing methods with the advantages of earlier detection of infection, high sensitivity and specificity [2, 3] . However, the results of RT-PCR nucleic acid detection are affected by multiple factors, such as the accuracy and reproducibility of the equipment, the easy degradation of stored and transported RNA, the duration of the patient's infection, etc. [4, 5] . This situation increases the risk of false negatives. It turns out that the probability of false negative is relatively high, which is very dangerous [6, 7] . This has also led to the suffering of all mankind.

As everyone knows that serum-specific antibodies are another key evidence for the diagnosis of infection as an important effector molecule of the body's immune system against viruses. The operation of antibody detection is simple and convenient, and it does not require high experimental environment and technical personnel. It only needs to collect blood samples, which can greatly reduce the risk of infection of medical staff during specimen collection and detection. At the same time, antibody testing can avoid the occurrence of false negatives [7, 8] . It is an important means to assist nucleic acid diagnosis. Currently, serum antibody testing is used as an auxiliary method to detect 2019-nCoV [7, [9] [10] [11] .

Fluorescence immunosensor has received widespread attention. It has many advantages as a substitute for conventional immunoassays, such as simple operation, fast response time, high specificity and sensitivity, and good practicability [12] . In recent years, nanocomposites have attracted much attention in the field of the fluorescence immunosensor. [13] [14] [15] [16] One kind of nanomaterial usually cannot meet all requirements. Therefore, the combination of two or more nanomaterials can meet more requirements and is more suitable for fluorescent immunosensor applications. Graphene quantum dot is a new type of quantum dot, which exhibits stronger boundary effects than graphene.

In addition, graphene quantum dots also exhibit low cytotoxicity, good water solubility, chemical inertness, stable photoluminescence, size tunable fluorescence signatures and other properties [17] [18] [19] [20] .

These properties make them attractive fluorescent labels for the sensing of biological assays. For instance, A novel dual-mode immunoassay was designed by Zou et al. [21] . The tuberculosis (TB) antigen CFP-10 is immunoassayed by using graphene quantum dots through fluorescence and surfaceenhanced Raman scattering (SERS). Ag@AuNPs, a bimetallic nanomaterial, not only combines the properties of a single metal element, but also has better performance than a single metal element. It enhances the stability and dispersion of the material, and improves the biocompatibility [22, 23] . In 2018, Kong et al. [24] designed a method to detect von Willebrand factor (vWF). This method uses a complex of GQD and Ag@AuNPs as an immunosensor.

In this work, Ag@AuNPs and GQDs were successfully synthesized. Ag@AuNPs can be used as 

GQDs stock solution was prepared from pyrolytic citric acid (CA) with minor modifications as reported in the literature [25] . 2 g CA was put into a beaker and heated at 200℃ for 30 min. CA gradually changed from a colorless solid to an orange liquid.Then, the orange liquid was slowly moved to NaOH (100 mL, 10 mg mlL -1 ) solution, while stirring to uniform the solution. Finally, the GQDs solution was obtained by adjusting the pH value of GQDs to 8.0, which was stored at 4 ℃.

Ag@AuNPs were carried out using a previous method [26] with minor modification. The total The Ag@AuNPs-NCP antigen (Ag@AuNPs-NCP) was prepared using the previous method [24] with slight modifications: First, under stirring conditions, NCP antigen (300μL, 50μg mL -1 ) was added to 3 mL Ag@AuNPs suspension. Then BSA solution (60μL, 1%) was added to block non-specific sites on the Ag@AuNPs-NCP surface. The above solution was continuously stirred at 4℃ for 4 h and centrifuged for 30 min (13,000 rpm). Finally, the sediment was ultrasonically redissolved in 1.5 mL PBS (pH 7.5).

All experiments were performed in PBS (pH 7.5). 100μL Ag@AuNPs-NCP and 20μL 2019-nCoV mAb of different concentration were added to a centrifuge tube to mix well, and incubated at room temperature for 150 min. Then, 20μL GQDs solution was added to the centrifuge tube. Finally, 160μL of PBS (pH 7.5) buffer solution was added to the above solution. The fluorescence spectrum of the solution was measured.

The artificial serum was obtained from Huzhou Inno Reagents Co., Ltd and diluted 100 times with PBS (pH 7.5) before sensing. The diluted serums were spiked with known concentrations of 2019-nCoV mAb and measured under the optimum conditions.

As shown by the TEM (Fig. 2A) , the diameter of GQD is 15-20nm. It is nearly spherical and has good dispersibility. This is similar to the reported literature [25] . The TEM image (Fig.2B) shows that Ag@AuNPs were spherical with an average grain diameter of 25 nm. This is consistent with reports in the literature [24] . The surface chemical composition of Ag@AuNPs was determined by XPS. The Ag3d spectrum can be seen in Fig. 2C . Because of the spin-orbit coupling, the binding energies are 367.7 and 373.7 eV. Its intensity ratio is 2:3, corresponding to Ag3d3/2 and Ag3d5/2 respectively. These are put down to the metallic silver in Au@Ag NPs [27] [28] [29] [30] . As plotted in Fig. 2D , there are two main peaks at 83.6eV and 87.3eV, corresponding to Au4f7/2 and Au4f5/2, respectively. This proves the existence of metallic gold. The observed position is consistent with those reported in literature [27] [28] [29] 31 ]. In addition, in order to research the optical properties of GQDs, the obtained GQDs were characterized by spectroscopy. The blue line in Fig. 2E is the UV-Vis spectrum of graphene quantum dots. The prepared GQDs show an obvious π-π* absorption peak at 365nm, which is related to the C=C transition in the UV-Vis spectrum [32] . At the same time, Ag@AuNPs have an obvious UV absorption peak near 490nm. The fluorescence emission spectrum of graphene quantum dots was given in Fig. 2F . When the excitation spectrum is 390 nm, the graphene quantum dots have a maximum emission spectrum at 465 nm. This is consistent with reports in the literature [33] , indicating that GQDs were successfully synthesized.

The first condition for FRET to occur is that there is a significant overlap between the absorption spectrum of the acceptor and the emission spectra of the donor. This allows the acceptor to absorb the energy released by the donor. Secondly, the fluorescent chromophores of the donor and acceptor must be arranged in an appropriate way, because the dipole has a certain spatial orientation. In addition, the donor and acceptor must be close enough, but the distance needs to exceed the collision diameter between them. In this case, FRET may occur [34] . The fluorescence emission peaks of GQDs can overlap with the absorption spectra of some receptors because their emission spectra are usually in the visible light region.

The fluorescence emission spectra of 2019-nCoV mAb detection is shown in Fig. 3 . It can be seen that GQDs (red line) have the highest fluorescence emission peak at 460 nm. In the presence of Ag@AuNPs, the fluorescence intensity was reduced to a certain extent. At the same time, GQDs have a strong UV absorption peak at 365 nm, while Ag@AuNPs have a significant peak at 490 nm, as given in the UV-Vis absorption spectrum (Fig. 2E ). In the figure, it can be clearly seen that the sum of the absorbance of GQDs and Ag@AuNPs (red line) is greater than the UV absorption peak of the mixture containing GQDs and Ag@AuNPs (green line), which indicates that there is an interaction between them. The fluorescence emission spectra of GQDs partially overlap with the UV absorption spectrum of Ag@AuNPs. The main conclusion that can be drawn is that there is FRET between them. [35] GQDs are donors and Ag@AuNPs are acceptors. It can be inferred from the above research that the fluorescence quenching of GDQs is based on the principle of FRET. It can be seen from the detection diagram that the immobilization of BSA and NCP antigen on Ag@AuNPs makes the distance between Ag@AuNPs-NCP and GQDs farther until the optimal distance for FRET is reached [36] . The FRET efficiency reaches maximum value, and the fluorescence intensity of GQDs is further reduced. When

Ag@AuNPs-NCP and 2019-nCoV mAb coexist, the distance between 2019-nCoV mAbnanocomposites and GQDs is further increased due to the steric-hinerance effect..At the same time, the efficiency of FRET decreases, and the fluorescence of GQDs turns on. 

In order to obtain excellent detection performance, the ratio of the concentration of Au to Ag in Ag@AuNPs, the ratio of GQDs to Ag@AuNPs, the reaction conditions of 2019-nCoV mAb and Ag@AuNPs-NCP were optimized, including pH and incubation time. According to the UV absorption spectrum of Ag@AuNPs with various concentration ratios of Au and Ag (Fig. S1) , it can be seen that when Au:Ag = 7:3, the overlap area of the UV absorption spectrum and the fluorescence emission spectrum of GQDs is the largest. And it has the best fluorescence quenching effect on GQDs (Fig. S2 ).

As shown in the Fig. S3 , Ag@AuNPs with a volume of 100μL were selected for the experiment. In addition, the influence of pH was studied in the range of pH 5.5-8.5 and was shown in Fig. 3B . The fluorescence intensity of GQDs containing Ag@AuNPs-NCP was denoted by F0. The fluorescence intensity of GQDs containing both 2019-nCoV mAb and Ag@AuNPs-NCP was represented by F1.

And their difference △F (△F = F0-F1) and the ratio of △F to F0 were calculated. When the pH values of the solution reach 7.5, the fluorescence enhancement of as-developed probe displays the maximum.

And the value of △F/F0 decreased under both acidic or alkaline conditions. According to previous literature report [37] , the stability of the biological activity of the immobilized protein is dependent on the pH of the solution. Acidic and alkaline solutions will reduce protein activity and cause significant decrease in the performance of the immunosensor. The effect of incubation time on the reaction system was shown in Fig. 3C . With the increase of incubation time, △F/F0 gradually increased and stabilized after 2.5 h. It can be seen that a longer incubation time will not improve the detection effect. Therefore, this study chose the optimal incubation time of Ag@AuNPs-NCP and 2019-nCoV as 2.5 h. ratio is 3, the detection limit is calculated to be 50 fg mL -1 .

In order to verify that the developed fluorescence immunosensor has good selectivity, potential coexisting components in serum are also tested for comparison. These coexisting ingredients include BSA (Bovine Serum Albumin), GSH (glutathione), L-cysteine, glucose, L-histidine, and folic acid.

The fluorescence intensity of GQDs containing Ag@AuNPs-NCP was detected. And on this basis, 2019-nCoV mAb (10 ng mL -1 ) and the above components (100 ng mL -1 ) were added separately for detection. As can be seen from Fig. 4A , the fluorescence intensity of the 2019-nCoV mAb fluorescent immunosensor based on the principle of FRET is greater than that of the negative control. Therefore, this system has good selectivity to 2019-nCoV mAb. Fig. 4C) , it can be seen that when the dilution times are 100 times, the fluorescence intensity of 2019-nCoV mAb in serum was closer to the fluorescence intensity in ultrapure water, and the recovery rate was better. Therefore, the serum was diluted 100 times and samples of standard 2019-nCoV mAb containing 1, 0.1, and 0.01ng mL -1 were prepared for testing. The recovery ranged from 90.8 % to 103.3 % with relative standard deviations (RSD) less than 10.3 % ( Table 1 ). The method showed good accuracy in the detection of 2019-nCoV mAb in artificial serum. 

A new type of 2019-nCoV antibody fluorescence immunoassay method combining GQDs and

Ag@AuNPs is proposed. This study prepared fluorescent GQDs and used them as fluorescent probes.

By analyzing its own peak and UV spectrum characterization, its feasibility was studied. The prepared 

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The author would like to thank for the financial support of the National Natural Science 

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: