key: cord-0896276-dcx33pja
authors: Guo, Jiaping; Wang, Yameng; Niu, Shanshan; Li, Hongping; Tian, Yongmei; Yu, Songcheng; Yu, Fei; Wu, Yongjun; Liu, Li-e
title: Highly Sensitive Fluorescence-Linked Immunosorbent Assay for the Determination of Human IgG in Serum Using Quantum Dot Nanobeads and Magnetic Fe(3)O(4) Nanospheres
date: 2020-08-31
journal: ACS Omega
DOI: 10.1021/acsomega.0c02987
sha: 2f285d121695893786d12f4f572bc9f7b58561cf
doc_id: 896276
cord_uid: dcx33pja

[Image: see text] The aim of this study is to establish a new method with high sensitivity, accuracy, and stability for the determination of human IgG and then expand it to analyze severe acute respiratory syndrome corona virus 2 (SARS-CoV-2)-specific IgM and IgG, which is of great significance for the screening and diagnosis of COVID-19. In this study, the magnetic Fe(3)O(4) nanospheres coupled with mouse antihuman IgG (Ab1(IgG)) were used as an immune capture probe (Fe(3)O(4)@Ab1(IgG)) to capture and separate the target, and rabbit antihuman IgG (Ab2(IgG)) coupled with highly luminescent quantum dot nanobeads (QBs) as a fluorescence detection probe (QBs@Ab2(IgG)) was used to realize high sensitivity detection. After the formation of a sandwich immunocomplex, the fluorescence intensity of the precipitate after magnetic separation was measured at the excitation wavelength of 370 nm. Under optimal conditions, a wide linear range varying from 0.005 to 40 ng·mL(–1) was obtained for the detection of human IgG with a lower limit of detection at 4 pg·mL(–1) (S/N = 3). The recoveries of intra- and interassays were 90.0–101.9 and 96.0–106.6%, respectively, and the relative standard deviations were 6.3–10.2 and 2.6–10.5%, respectively. Furthermore, the proposed method was successfully demonstrated to detect human IgG in serum samples, and the detection results were not statistically different (P > 0.05) from commercial enzyme-linked immunosorbent assay kits. This method is sensitive, fast, and accurate, which could be expanded to detect the specific IgM and IgG antibodies against SARS-CoV-2.

In December 2019, a kind of pneumonia infected by a novel coronavirus broke out in Wuhan, Hubei, China, which rapidly spread and seriously threatened the health and life safety of the people. On 11 February 2020, the World Health Organization (WHO) officially named the new coronavirus pneumonia as coronavirus disease 2019 (COVID- 19) . 1 On the same day, the Coronavirus Study Group of the International Committee on Taxonomy of Viruses named the virus as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 2 As of 19 June 2020, the total number of confirmed cases worldwide has reached 8,385,440, and the death toll has exceeded 450,686. 3 More worryingly, specific drugs and vaccines will not be available soon. 4−6 Therefore, rapid screening of patients and asymptomatic cases is still the key to the prevention and control of COVID-19.

Although the positive result of SARS-CoV-2 nucleic acid test is the "gold standard" for the diagnosis of COVID-19, there is a certain proportion of false negative because of many factors, such as sample type, quality, delivery time, and so on. 7, 8 Serological testing is another major screening technique for the asymptomatic cases, previous infections, and their close contacts. 9, 10 In addition to the role of alternative or complementary methods to confirm suspected cases, this detection method will also provide important information about the human immune response process and be used to evaluate the effectiveness of the vaccine. 11 At present, enzymelinked immunosorbent assay (ELISA) 12, 13 and flow immunoassay 14−16 are the two main methods to detect specific antibodies against SARS-CoV-2. Although these methods are simple and convenient, the sensitivity and accuracy need to be further improved, especially in some special cases such as proofreading and verification. In view of their defects, chemiluminescent immunoassay (CLIA), 17,18 single molecule array assay, 19 indirect immunofluorescence assay, 20 and other techniques have been gradually developed, so it is necessary to constantly improve and innovate the existing methods. Theoretically, IgM appeared earlier in serum than IgG but because of its lower content, shorter duration, lower sensitivity, and specificity, some studies 21, 22 have shown that serum IgG amounts can increase at the same time or earlier than those of IgM against SARS-CoV-2. Therefore, it is of great significance to establish a highly sensitive, accurate, and stable method for the detection of human IgG and then expand it for the detection of SARS-CoV-2-specific IgM and IgG.

Magnetic Fe 3 O 4 nanospheres have the advantages of uniform particle size distribution, large specific surface area, easy modification, good water solubility, strong dispersion, and excellent magnetism, so it is an ideal carrier material with good separation effect. 23−26 Quantum dot is an excellent fluorescent material, which has the characteristics of broad excitation range, narrow emission spectra, high fluorescent quantum yield, large molar extinction coefficient, and superior brightness and durability to photobleaching. 27−31 Furthermore, because of the doping of a large number of quantum dots, the stability and fluorescence intensity of quantum dot nanobeads (QBs) are significantly higher than those of quantum dots, which can effectively improve the sensitivity of the detection method. 32−34 Thus, it would be a good choice to establish a fluorescence-linked immunosorbent assay (FLISA) based on magnetic Fe 3 O 4 nanospheres and QBs to detect human IgG in serum.

In this design, an immune capture probe was prepared by using 1-(3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride/N-hydroxysulfosuccinimide (EDC/Sulfo-NHS) as a coupling agent to conjugate mouse antihuman IgG (Ab1 IgG ) on the surface of carboxylated magnetic Fe 3 O 4 nanospheres. The IgG fluorescence detection probe was prepared by coupling rabbit antihuman IgG (Ab2 IgG ) to the surface of QBs by the same method. When human IgG is present, Fe 3 O 4 @Ab1 IgG , human IgG, and QBs@Ab2 IgG would form a sandwich immunocomplex, and the precipitation has a strong fluorescence signal after magnetic separation. When human IgG is absent, the fluorescence detection probe was discarded after magnetic separation, and the precipitate does not show fluorescence signals. Moreover, the practicability and accuracy of the sensor have been successfully confirmed in the detection of human IgG in serum. Therefore, the assay established in this study has a potential application prospect in the IgG detection of complex biological samples and can provide some inspiration and theoretical basis for the establishment and application of SARS-CoV-2 serum antibody detection technology.

2.1. Principle of FLISA. In this work, Ab1 IgG and Ab2 IgG were successfully coupled to magnetic Fe 3 O 4 nanospheres and QBs under the action of EDC/Sulfo-NHS, respectively. As depicted in Scheme 1, after the prepared Fe 3 O 4 @Ab1 IgG reacted with the human IgG standards or samples for a period of time, the targets were captured and enriched by the specific binding of antibody and antigen, and then the unbound standards or samples were removed by magnetic separation. When QBs@Ab2 IgG was added, the QBs@Ab2 IgG would bind to human IgG specifically by antigen−antibody reaction so that the Fe 3 O 4 @Ab1 IgG −human IgG−QBs@Ab2 IgG formed a sandwich structure, and the excess QBs@Ab2 IgG was washed away by magnetic separation. The fluorescence intensity of the reaction product was measured under the excitation wavelength of 370 nm, and the fluorescence intensity was positively correlated with the target concentration.

2.2. Characterization of Fe 3 O 4 @Ab1 IgG and QBs@ Ab2 IgG . The magnetic Fe 3 O 4 nanosphere, Fe 3 O 4 @Ab1 IgG , and QBs were characterized by transmission electron microscopy (TEM). As shown in Figure 1 , compared with magnetic Fe 3 O 4 nanospheres (a), the surface of Fe 3 O 4 @Ab1 IgG (b) has changed, which can preliminarily judge the coupling success of Fe 3 O 4 and Ab1 IgG . The particle size distribution of QBs was relatively uniform, and the surface of the nanospheres was coated with quantum dots (Figure 1c ).

The combination of carboxylated Fe 3 O 4 with Ab1 IgG was confirmed by hydrodynamic diameter and zeta potential. The hydrodynamic diameter of Ab1 IgG -modified Fe 3 O 4 increased ( Figure S1a ) and the zeta potential decreased ( Figure S1b ), suggesting that Fe 3 O 4 @Ab1 IgG has been successfully prepared. Moreover, the infrared spectrum of Fe 3 O 4 @Ab1 IgG ( Figure  S2a ) has one more characteristic absorption peak than that of Fe 3 O 4 ( Figure S2b ). In addition, the biological activity of Fe 3 O 4 @Ab1 IgG was determined by ELISA. First, Fe 3 O 4 @ Ab1 IgG was used to react with human IgG and Ab2 IgG -HRP. After adding 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution and incubating for 20 min, H 2 SO 4 solution was put in to stop the reaction. Finally, the optical density (OD) value was measured at 450 nm. It can be seen from Figure 1d that OD value increased with the increase of human IgG concentration and declined with the capture probe dilution ratio, indicating that Fe 3 O 4 @Ab1 IgG still possessed immunoreactivity to human IgG.

Similarly, we also examined the properties of QBs@Ab2 IgG . Compared with QB, the hydrodynamic diameter of Ab2 IgGmodified QBs increased ( Figure S1c ) and the zeta potential decreased ( Figure S1d ), indicating that the conjugate QBs@ Ab2 IgG was successfully prepared. At the same time, the addition of a new characteristic absorption peak in the infrared spectrum also confirmed the formation of QBs@Ab2 IgG ( Figure S2cc ). In order to obtain more accurate and reliable fluorescence signals, the excitation wavelength was optimized to 370 nm ( Figure S3 ). Next, we also evaluated the biological ACS Omega http://pubs.acs.org/journal/acsodf Article activity of QBs@Ab2 IgG by ELISA. As can be seen from Figure  1e , the changing trend of OD value was closely related to the concentration of human IgG and the dilution ratio of QBs@ Ab2 IgG , which indicated that Ab2 IgG bound with QBs still retained immunoreactivity.

Conditions. Immune capture probe and fluorescence detection probe, as the intermediate materials of the experimental process, played an important role in the whole experiment. Therefore, in order to improve the quality of the capture probe and save the cost and reaction time (Figure 2a) , the amount of antibody (Figure 2b) , the amount of coupling agent ( Figure S4a ), buffer solution ( Figure S4b ), and reaction temperature ( Figure S4c) were optimized with the coupling efficiency (the calculation method is shown in the Supporting Information) as the index. The results showed that the optimal immune capture probe was obtained by adding 1.0 mg of EDC, 2.0 mg of Sulfo-NHS, 10 μg of Ab1 IgG and reacting in phosphate buffer saline (PBS) solution at 25°C for 2 h. Similarly, in order to synthesize the high-quality fluorescence detection probe, the amounts of Ab2 IgG , Sulfo-NHS and EDC were optimized to 30 μg, 20 mg, and 5 mg, respectively (Table S1 ).

In addition, the sensitivity of the whole experiment mainly depended on the concentration of the immune capture probe and fluorescence detection probe, so the dilution ratio of Fe 3 O 4 @Ab1 IgG and QBs@Ab2 IgG was optimized. According to Figure 2c ,d, the fluorescence intensity increased at first and then decreased with the decrease of probe concentration. Under the condition of high concentration, the fluorescence intensity weakened rapidly. The reason may be that the high concentration of Fe 3 O 4 @Ab1 IgG caused the aggregation phenomenon 35−37 or the color of Fe 3 O 4 @Ab1 IgG (black) interfered with the fluorescence of the immunocomplex. 38, 39 Similarly, the phenomenon of fluorescence weakening caused by excessive concentration of signal detection probe is consistent with other reports. 37,40−42 Therefore, the optimal dilution ratios of Fe 3 O 4 @Ab1 IgG and QBs@Ab2 IgG were 1:40 and 1:4, respectively.

2.4. Analytical Performance of the Proposed Assay. Under the optimal conditions, the analytical performance of the FLISA was explored for the determination of human IgG. The calibration plot of human IgG at different concentrations is shown in Figure 3a , from which we could see that the fluorescence intensity increased gradually with the increase of IgG concentration in a certain range. This method showed a good linear relationship between the relative fluorescence units (RFU) and human IgG in the concentration range of 0.005− 40 ng·mL −1 , the linear regression equation was Y = 757.498 + 141.33X (where X denotes lg C IgG , and Y denotes RFU), the correlation coefficient (r) was 0.9946, and the limit of detection (LOD) was 0.004 ng·mL −1 (S/N = 3). Then, human IgG, human IgM, and human IgA were detected at the same concentration (25 ng·mL −1 ). The results clearly showed that the fluorescence response of human IgG was much greater than that of the other two antigens (Figure 3b ), indicating that this method has high selective recognition ability for human IgG. Finally, the accuracy and precision were verified by quantitative analysis of serum spiked samples with different concentrations (0.05, 1, 25 ng·mL −1 ) of IgG. The recoveries of intra-and interassays were 90.0−101.9 and 96.0−106.6%, respectively; and the relative standard deviations were 6.3− 10.2 and 2.6−10.5%, respectively.

Furthermore, the method established in this study was compared with the recently reported optical methods for the analysis of IgG. We can see from Table 1 that the proposed FLISA has a wider linear range and a lower LOD than others. In conclusion, this method is an accurate, sensitive, low-cost, time-saving, and high-throughput method for the detection of IgG, which has a great potential value in scientific research and clinical practice.

2.5. Detection of Human Serum Samples. In order to further confirm the applicability of this method to complex biological samples, 20 serum samples diluted 50,000 times were detected by FLISA, and the results were compared with (Figure 4) , and the original data are shown in Table S2 .

In summary, we designed a new fluorescence immunoassay method by combining nanotechnology, magnetic separation technology, and immunoassay technology and proved that it can be applied to detect human IgG in serum samples. Because of its large specific surface area, the immobilization of antibodies with magnetic Fe 3 O 4 nanoparticles can overcome the shortcomings of conventional ELISA analysis, such as easy shedding of coated antigen (or antibody), small solid−liquid contact area, long analysis time, and so on. In addition, as a high fluorescence material, QBs significantly improved the sensitivity of the experiment. As a result, in this study, FLISA had a wide linear range, high sensitivity, high accuracy, good precision, and simple and quick operation and showed better performance than traditional analytical methods. Besides, the experiment was carried out in a 96-well plate, which provided a possibility for high-throughput and large-sample detection. In addition, the detection of other antigens can be realized only by replacing the corresponding antibodies, and the simultaneous determination of multicomponents can also be achieved by adding a variety of fluorescent materials with different emission wavelengths. Therefore, this study can be conceivably expanded to detect SARS-CoV-2-specific IgM and IgG in serum and used for the rapid screening of infected patients and asymptomatic carriers. Although the flow immunoassay is simple, convenient, fast, and widely used in various fields, 49−53 it is necessary to further improve the sensitivity in the detection of SARS-CoV-2-specific antibodies. 54 The higher the sensitivity, the earlier the specific antibodies can be detected, that is, the earlier measures can be taken for prevention, diagnosis, and treatment. Therefore, the methods with high sensitivity, high accuracy, and good repeatability established in this study can complement each other with flow immunoassay, especially in the fields of proofreading or verifying data and scientific research. Fluorescence, UV−vis, and IR spectra were recorded with an F-4500 spectrofluorophotometer (HITACHI Co., Ltd., Japan), a UV-1601 UV−vis spectrometer (Shimadzu Co., Ltd., Japan), and a PE-1710 IR spectrometer (West PE Co., Ltd., Germany), respectively. RFU and OD were measured by a multimode microplate reader SpectraMax M2e (USA). TEM images were recorded using a JEM-2100 transmission electron microscope (Japan). Hydrodynamic diameter and zeta potential were analyzed using a Zetasizer Nano-zs 90(Malvern Co., Ltd., England). A magnetic panel was used for magnetic separation (Baiyun Nano Technology Co., Ltd., China).

4.2. Preparation of an Immune Capture Probe (Fe 3 O 4 @Ab1 IgG ) and a Fluorescence Detection Probe (QBs@Ab2 IgG ). The Fe 3 O 4 @Ab1 IgG conjugates were synthesized by an EDC/Sulfo-NHS chemical bonding method. At first, 100 μL of 50 mg·mL −1 carboxylated magnetic Fe 3 O 4 nanospheres was washed three times with 300 μL of PBS (pH 7.4, containing 0.01 mol·L −1 PBS). Subsequently, 100 μL of 0.1 mg·mL −1 Ab1 IgG was added and gently stirred to mix. After that, 100 μL of EDC/Sulfo-NHS (10 mg·mL −1 EDC and 20 mg·mL −1 Sulfo-NHS were prepared by PBS) was put together and the mixture was incubated at 25°C for 2 h, followed by magnetic separation and washing twice with PBS. Afterward, in order to block the unreacted activated groups, 300 μL of blocking buffer (pH 7.4, containing 0.01 mol·L −1 PBS and 0.5% BSA) was injected into the tube with stirring for 30 min and then washed twice with 300 μL of PBS. After being mixed with 300 μL of PBS, the final tubes were stored at 4°C. In a similar strategy, QBs were coupled with Ab2 IgG to prepare fluorescence detection probes. Blood samples were taken from the First Affiliated Hospital of Zhengzhou University. About 3 mL of fasting venous blood was placed in a nonanticoagulant tube and then kept in a water bath at 37°C for 30 min. After that, the serum was centrifuged at the speed of 3000 rpm for 5 min to separate the serum and then placed in a dry freezer and stored at −80°C until use.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02987.

Hydrodynamic diameter, zeta potential, and infrared spectrum characterization of Fe 3 O 4 @Ab1 IgG and QBs@ Ab2 IgG ; selection of the best excitation wavelength; calculation method of coupling efficiency; optimization of Fe 3 O 4 @Ab1 IgG and QBs@Ab2 IgG preparation; and detection results of human serum by using FLISA and a commercial ELISA kit (PDF)

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