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. 2023 Mar 20;1255:341102. doi: 10.1016/j.aca.2023.341102

Gold-silver alloy hollow nanoshells-based lateral flow immunoassay for colorimetric, photothermal, and SERS tri-mode detection of SARS-CoV-2 neutralizing antibody

Tianyu Zhao a,1, Penghui Liang a,1, Jiaqi Ren a, Jinyue Zhu a, Xianning Yang a, Hongyu Bian a, Jingwen Li a, Xiaofeng Cui b, Chunhui Fu b, Jinyan Xing c,∗∗, Congying Wen a,∗∗∗, Jingbin Zeng a,∗
PMCID: PMC10026621  PMID: 37032051

Abstract

Although many approaches have been developed for the quick assessment of SARS-CoV-2 infection, few of them are devoted to the detection of the neutralizing antibody, which is essential for assessing the effectiveness of vaccines. Herein, we developed a tri-mode lateral flow immunoassay (LFIA) platform based on gold–silver alloy hollow nanoshells (Au–Ag HNSs) for the sensitive and accurate quantification of neutralizing antibodies. By tuning the shell-to-core ratio, the surface plasmon resonance (SPR) absorption band of the Au–Ag HNSs is located within the near infrared (NIR) region, endowing them with an excellent photothermal effect under the irradiation of optical maser at 808 nm. Further, the Raman reporter molecule 4-mercaptobenzoic acid (MBA) was immobilized on the gold–silver alloy nanoshell to obtain an enhanced SERS signal. Thus, these Au–Ag HNSs could provide colorimetric, photothermal and SERS signals, with which, tri-mode strips for SARS-CoV-2 neutralizing antibody detection were constructed by competitive immunoassay. Since these three kinds of signals could complement one another, a more accurate detection was achieved. The tri-mode LFIA achieved a quantitative detection with detection limit of 20 ng/mL. Moreover, it also successfully detected the serum samples from 98 vaccinated volunteers with 79 positive results, exhibiting great application value in neutralizing antibody detection.

Keywords: SARS-CoV-2 neutralizing antibody, Colorimetry, Surface-enhanced Raman scattering, Photothermal effects, Lateral flow immunoassay

Graphical abstract

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1. Introduction

Since 2019, Corona Virus Disease 2019 (COVID-19) has spread rapidly around the world, which has placed a severe burden on the global health care system and economy [1]. According to data published by the World Health Organization (WHO), as of September 2022, the cumulative number of confirmed cases has exceeded 607 million, with more than 6.49 million deaths and a progressive increase in the number of infections [2]. Vaccination is by far the most effective medical intervention to stop an outbreak in its tracks and help us get back on track [3]. Vaccination induces the production of neutralizing antibodies that effectively prevent infection and protect the body [[4], [5], [6], [7]]. Neutralizing antibody detection is important for vaccine development, application, and the understanding of the epidemiological background and changes of SARS-CoV-2.

Testing for viral neutralization is currently the gold standard for detecting SARS-CoV-2 neutralizing antibodies [[8], [9], [10], [11]]. In spite of this, it entails a very technical and time-consuming process as well as a high level of risk. In order to conduct live virus and pseudovirus neutralization testing, biosafety level 3 and 2 facilities must be equipped [12]. Principle of testing neutralizing antibodies against SARS-CoV-2 based on the evaluation of their ability to inhibit binding between the SARS-CoV-2 S protein RBD and the angiotensin converting enzyme 2 (ACE2) protein, the enzyme-linked immunosorbent assay (ELISA) [13,14] and plasmon resonance assay (SPR) [15,16] have recently been developed for the purpose of neutralizing the SARS-CoV-2 virus. However, their reliance on large equipment and operators, as well as their time-consuming and labor-intensive operation, limit their widespread use. For this reason, immediate immunosensors that can detect SARS-CoV-2 neutralizing antibodies urgently need to be developed, routinely available, and rapid.

Recently, lateral flow immunoassay (LFIA) has gained much attention because of its simplicity, rapid detection, low cost, and the ability to perform on-site testing free of large equipment [17,18]. In the wake of these developments, the LFIA technique is now widely used as a tool to determine pesticides [[19], [20], [21]], mycotoxins [22,23], pathogens [24,25], as well as disease biomarkers, such as SARS-CoV-2 antigens and antibodies [26,27]. And, self-testing for COVID-19 using LFIA is now widely used. However, conventional LFIA is limited by low sensitivity and the inability to quantify due to the use of Au nanoparticles (NPs) as reporters, which may lead to misinterpretation of results. Several nanobeacons have been developed over the past few years by a variety of researchers to replace colloidal gold. For example, magnetic nanoparticles [28], fluorescent microspheres [29], plasmonic nanoparticles [30], and many other types of nanoparticles have been developed. In recent years, the photothermal activity of nanomaterials have been exploited in LFIA detection. Photothermal signals can be interpreted with simple thermometers or infrared cameras, which, in addition to reducing the need for more complicated equipment, increases the chances of a successful detection by improving the sensitivity of the equipment [31,32]. Additionally, surface-enhanced Raman scattering (SERS) nanotags have been integrated with the LFIA platform to create ultrasensitive indicators for trace biological compounds. SERS nanotags may provide powerful fingerprint signals [33,34]. In our earlier research, we combined Ag nanoparticles with very thin Au shells (2 nm) coated with 4-mercaptobenzoic acid (MBA) into LFIA for highly sensitive dual-mode colorimetric and SERS detection of SARS-CoV-2 IgG.

In this study, we developed gold–silver alloy hollow nanoshells (Au–Ag HNSs) as novel reporters for colorimetric, photothermal, and SERS tri-mode detection of SARS-CoV-2 neutralizing antibodies, drawing on pioneer research and our earlier work as inspiration. The Au–Ag HNSs were prepared by template etching using Ag NPs. Their Ultraviolet–visible (UV–vis) extinction peak was at 720 nm, which made them have an excellent photothermal effect. Further, the SERS reporter molecule MBA was immobilized on the gold–silver alloy nanoshell to obtain an enhanced SERS signal. The alloy structure endowed Au–Ag HNSs with the surface chemistry advantages of Au and superior optical characteristics of Ag. Thus, Au–Ag HNSs had higher stability and biocompatibility than Ag NPs, and showed superior SERS performance than a traditional gold shells [35]. These Au–Ag HNSs could provide colorimetric, photothermal, and SERS signals. The technology allowed for quantitative photothermal and SERS detection in the range of 20–1500 ng/mL as well as naked-eye qualitative detection with a limit of 160 ng/mL. Due to the mutual complementarity of these three signals, the accuracy of the detection would be well improved. Finally, this tri-mode LFIA was successfully detected the serum samples from 98 vaccinated volunteers with 79 positive results, exhibiting great application value in neutralizing antibody detection. Compared with similar work for SARS-CoV-2 IgG [26,36] and neutralizing antibody detection [37,38], our method allows for photothermal, colorimetric and SERS tri-modal detection with higher sensitivity and accuracy.

2. Experimental section

2.1. Materials, reagents, and instruments

From Shanghai Hushi Chemical Reagent Co., Ltd., sodium borohydride (NaBH4) and trisodium citrate (TSC) were acquired. Macklin supplied the polyvinyl pyrrolidone (PVP, MW 10000). Energy-chemical Co., Ltd. provided chloroauric acid (HAuCl4), sucrose, MBA, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) (Shanghai, China). SH-PEG-COOH (MW = 2000) was obtained from ToYongBio Tech. Inc. (Shanghai, China). Sino Biological Inc. supplied the SARS-CoV-2 Spike protein, SARS-CoV-2 ACE2 protein, SARS-CoV-2 Spike antibody, and SARS-CoV-2 neutralizing antibody (Beijing, China). Sigma-Aldrich was used to obtain mouse anti-Staphylococcus aureus and anti-Salmonella typhimurium antibodies (Saint Louis, MO, U.S.A.). The company EKEAR Bio@Tech Co., Ltd. sold Tween-20 (Shanghai, China). Nitrocellulose membrane (NC membrane), glass fiber conjugate pad, absorbent pad, and PVC substrate were obtained from Joey-biotech Co., Ltd. (Shanghai, China). The Sartorius CN140 nitrocellulose membrane was bought from Sartorius in Göttingen, Germany. Serum samples were obtained from Qingdao University's associated hospital and kept there for usage at −20 °C.

An Autopure WR600A system from Millipore was used to get ultrapure water (18.2 MΩ·cm). On a UV-2450 spectrophotometer, the UV–vis absorption spectra were measured (Shimadzu). Pictures were taken using an iPhone12 (Apple). Using a JEM 1400 microscope at 120 kV operating voltage, transmission electron microscopy (TEM) pictures were taken (JEOL). The SR-510PRO Raman analyzer was used to measure the SERS scattering spectra (Ocean optics). Thermal camera Fotric 226s was used for photothermal imaging. From Fulei Tech Co., Ltd., an 808 nm excitation laser with power of 2 W/cm2 laser light was acquired (Shenzhen, China). The HGS510 (AUTOKUN) sprayer and the HGS210 induction cutting machine were used to separate the test strips (AUTOKUN).

2.2. Preparation of Au–Ag HNSs

The synthesis of Ag nanoparticles was performed in a similar manner to the previous article [26]. The Au shells were synthesized by using Ag NPs as etching templates. A mixture of 5 mL of Ag NPs and 45 mL of 0.1% (w/v) PVP was heated to 100 °C with magnetic stirring. The solution was then gradually supplemented with 1 mM HAuCl4 at a rate of 300 mL/min. The process was halted as soon as the hue of the solution became blue and the extinction peak's wavelength reached 720 nm. The resulting Au–Ag HNSs were dispersed in 5 mL of water after being centrifuged and rinsed with ultrapure water. Au–Ag HNSs were treated with 100 μL of a 1 mM MBA solution, and the mixture was vigorously stirred for 1 h. By centrifuging out the free MBA, the resulting Au–Ag HNSs were then re-dispersed in 10 mL of water. As shown in Fig. 1 a, the entire synthesis and functionalization process was depicted throughout the process.

Fig. 1.

Fig. 1

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(a) Flowchart showing the steps involved in synthesizing and functionalizing Au–Ag HNSs. (b) Diagram of the detection principle of the tri-mode assay and test strip for detecting SARS-CoV-2 neutralizing antibody using Au–Ag HNSs.

2.3. Functionalization of the Au–Ag HNSs with SARS-CoV-2 S protein

2.5 mL of 4.0 μg/μL SH-PEG-COOH solution and 10 mL of Au–Ag HNSs were blended, and the mixture was vigorously stirred at room temperature for 1 h. Au–Ag HNSs were then centrifuged three times for washing before being dissolved in 1.0 mL of PBS (0.01 M pH = 7.2). The aforementioned solution received 25 μL of 40 μg/μL EDC and 25 μL of 20 μg/μL NHS in that order, and for 30 min, it was moderately shaken at room temperature. The next step was to wash Au–Ag HNSs twice and dissolve with 10 μg of SARS-CoV-2 S protein in a solution of PBS (0.01 M pH = 7.2). The reaction was performed for 2 h. For further analysis, the immunonanoshells (INSs) were stored at 4 °C after washing with PBS, blocking with 5% BSA, and dispersing in 0.01 M pH = 7.2 PBS.

2.4. Preparation of the LFIA strip

The NC membrane, the sample pad, the conjugate pad, and the absorbent pad of the test strip were pasted to the PVC substrate in the order shown in Fig. 1b. SARS-CoV-2 S protein antibody was sprayed at a rate of 1.0 μL/cm onto control (C) lines, while ACE2 protein was sprayed at a rate of 0.8 μL/cm onto test (T) lines [39,40]. The LFIA test strips had to be dried at 37 °C for a total of 12 h before they could be used again. Then they were cut into 3 mm wide strips. These were stored at a temperature of 25 °C for storage. The last step before using the test strip was to add 100 μL of resuspension solution (0.5% Tween-20 and 1% BSA in pH = 7.2 PBS) to the sample pad to block the test strip and then let it dry at room temperature for several hours before using.

2.5. Detection of SARS-CoV-2 neutralizing antibody

It was therefore decided to carry out the tri-mode LFIA analysis as follows in order to analyse the neutralizing antibody against SARS-CoV-2. In order to create the microtiter plate, 20 μL of the SARS-CoV-2 neutralizing antibody sample and a certain number of INSs were combined. The running solution (1.5% Tween-20, 1% BSA in pH = 7.2 PBS) was then successively added to create a total volume of 50 μL. LFIA test strip sample pads were treated with the above mixture dropwise. Afterwards, the strip was rinsed off running solution. Qualitative detection was made after 15 min by observing the color of the T line. In the image shown above, it can be seen that the LFIA test strip was irradiated with an 808 nm laser with a power of 2 W/cm2. During this period, a thermal camera of the type Fotric 226s was used to capture this picture. Temperature contrast (ΔT = ΔT1 - ΔT0), where ΔT1 and ΔT0 stand for the temperature change values before and after irradiation of the T line in the sample group and the blank (unspiked) group, respectively, was used to define the photothermal signal. As a final measurement, a portable Raman spectrometer with a 785 nm laser was used to measure Raman peak intensity at 1075 cm−1. The above photothermal and Raman signals both could be used for quantitative detection.

2.6. Application to clinical specimens and synthetic serum samples

Several concentrations of neutralizing antibodies against SARS-CoV-2 were injected into the serum of healthy volunteers to generate simulated serum samples. The samples were diluted 10 times with PBS and then tested with tri-mode LFIA following the procedure described. From clinical samples, blood taken form a total of 98 vaccinated and 9 unvaccinated volunteers were kept overnight at 2–8 °C and then centrifuged at 1000 g for 20 min at 2-8 °C before detection.

3. Results and discussion

3.1. Principle of tri-mode LFIA for the detection of SARS-CoV-2 neutralizing antibody

The tri-mode strips were constructed by indirect competitive immunoassay as shown in Fig. 1b. When the tested sample was negative, the SARS-CoV-2 S protein on INSs would react with the ACE2 protein fixed on the T line, and the INSs were trapped and aggregated on the T line, inducing a blue band. On the contrary, when the tested samples were positive, the SARS-CoV-2 neutralizing antibody in the sample would first competitively bind with the INSs, thus preventing the binding between the INSs and the ACE2 protein on the T line, which induced the T line color faded. With increasing the concentration of SARS-CoV-2 neutralizing antibody in the sample, fewer INSs would be trapped by the T line, and the color of the T line would be lighter. Meanwhile, the SARS-CoV-2 S protein on INSs reacted with the S protein antibody on the C line to form a colored band for quality control regardless of whether there was a SARS-CoV-2 neutralizing antibody in the sample or not. A qualitative analysis was achieved through visual inspection of the T line's color, whereas a quantitative analysis was achieved through the measurement of photothermal and SERS signals.

3.2. Characterization of Au–Ag HNSs

Au–Ag HNSs were prepared by template etching, which were grown on the surface of Ag NPs by etching them with HAuCl4 to form a shell layer. As shown in Fig. 2 a and b, the diameter of Au–Ag HNSs was about 35 nm, and the shell thickness of the prepared Au–Ag HNSs was about 6.1 nm. The composition of the nanoparticles was confirmed to be a gold-silver alloy. As shown in Fig. 2c,e,f, gold and silver elements were distributed on the layers. The EDX line scan of Au–Ag HNS (Fig. 2d) also showed that gold and silver elements were both distributed on the shell layer. EDS analysis (Fig. S1) also agreed with the above experiment results. Their UV–Vis absorption spectra showed that the maximum absorption peak was located at 720 nm when the molar ratio of added silver to gold reached 15:4 (Fig. 3 a). In addition, we compared the photothermal and SERS performance of Au–Ag HNSs with different UV absorption peaks, which showed different photothermal and SERS performance. As shown in Fig. 3b and c, the Au–Ag HNSs at a molar ratio of 15:4 had the highest photothermal effect and were significantly higher than the other Au–Ag HNSs. While the Au–Ag HNSs at a molar ratio of 15:1 achieved the strongest SERS performance but were comparable with the Au–Ag HNSs at a molar ratio of 15:4. On balance, we chose the Au–Ag HNSs at a molar ratio of 15:4 as the marker for the lateral flow immunoassay.

Fig. 2.

Fig. 2

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(a)–(b) TEM image of Au–Ag HNSs (Inset shows the distribution histogram of the thickness of Au–Ag HNSs). (c) STEM elemental maps of Au. (d) EDX line scan image of Au–Ag HNSs. The image of the line scan area is shown in the inset. (e) STEM elemental maps of Ag. (f) STEM elemental maps of Au and Ag overlay.

Fig. 3.

Fig. 3

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(a) UV–Vis extinction spectra of Au–Ag HNSs under different mole ratios. (b) Photothermal effects of Au–Ag HNSs under different mole ratios. (c) SERS spectra of Au–Ag HNSs under different mole ratios. The black, purple, red and blue line represents a silver-gold molar ratio of 15:0, 15:9, 15:1 and t 15:4, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.3. Tri-mode LFIA's analytical capabilities for detecting SARS-CoV-2 neutralizing antibodies

To obtain the optimal detection conditions, we optimized the amount of S protein coupled with Au–Ag HNSs, the amount of Au–Ag HNSs, and the incubation time of Au–Ag HNSs with the sample (Figs. S2a–c). In the final step, 1 μL of S protein, 15 μL of INSs, and 15 min of incubation were selected. This method was evaluated in serum samples under optimal conditions in order to determine its sensitivity and dynamic range. According to Fig. 4 a, both T and C lines were visible under natural light when there was no neutralizing antibody against SARS-CoV-2. When anti-neutralizing antibodies were 0.1 μg/mL in concentration, the color faded visually. With further increasing the neutralizing antibody concentration, the color of the T line became lighter due to the fact that more neutralizing antibodies bound with the Au–Ag HNSs which prevented the binding between the Au–Ag HNSs and the ACE2 protein on the T line. Therefore, the sample might be qualitatively and semi-quantitatively identified by examining the T line's color change. The photothermal and SERS signals of the T line were monitored for precise quantification. In the following Fig. 4c and d, the photothermal and SERS signals gradually decreased with increasing neutralizing antibody concentration. We introduce inhibition rate as a quantitative parameter, which is defined as follows

inhibition rate (%) = (S0-Ss)/S0 × 100%

Where S0 and SS are the signals obtained from the negative groups and sample groups. As shown in Fig. 4d, the photothermal and SERS signal both exhibited an exponential relationship versus the neutralizing antibody concentration from 20 to 1500 ng/mL in serum (R > 0.99), based on which, quantitative detection was realized. Photothermal and SERS measurements were calculated to have a limit of detection (LOD) of 20 ng/mL based on the lowest distinguishable signal (yblank + 3 × SDblank, where yblank is the average signal strength of the blank groups, and SDblank is the standard deviation of the blank measurements), which was ten times lower than the visual detection.

Fig. 4.

Fig. 4

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(a) Photographs of test strips for detecting various concentrations of SARS-CoV-2 neutralizing antibodies in serum. (b) Photographs of test strips for detecting various concentrations of SARS-CoV-2 neutralizing antibodies in serum taken by the thermal infrared imager. (c) SERS spectra obtained from the detection of SARS-CoV-2 neutralizing antibodies at various concentrations. (d) Relationship curve of photothermal and SERS signals versus neutralizing antibody concentration from 20 to 1500 ng/mL in serum. The error bars represented standard deviations calculated from three experiments.

3.4. Specificity and repeatability

Our further exploration was carried out to determine whether this method was specific and repeatable. To investigate the specificity of this approach, we tested a number of common proteins (Fig. 5 ). A strong signal could be generated by the quantitative detection of SARS-CoV-2 neutralizing antibodies by both photothermal and Raman techniques. With concentrations even 10-20 times higher than those of the blank samples, other proteins (SARS-CoV-2 N protein, SARS-CoV-2 N protein antibody, S. typhimurium antibody, and S. aureus antibody) did not produce evident signals.

Fig. 5.

Fig. 5

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Histograms of inhibition rates were generated using SERS and photothermal detection from serum samples spiked with various proteins.

This result showed that the method was highly specific, which led to the conclusion that the method was accurate. We also examined the coefficients of variation (CV) of the intra-assay and inter-assay experiments in serum under the photothermal mode as well as SERS mode, to ascertain whether this method was reliable. As can be seen in Tables S1 and 2, the intra-assay CVs were computed using the same batch of INSs to be 8.2 and 8.7%, respectively, while the inter-assay CVs were calculated using separate batches of Au–Ag HNSs to be 10.2 and 10.8%, respectively. It was found that both the photothermal and SERS modes of this method were reproducible, indicating that this method had good reproducibility under both modes.

3.5. Analyses of clinical samples

The technique was used to examine serum samples from 98 volunteers who received inactivated virus vaccine and 9 who had not received the vaccine. The results were shown in Fig. 6 that the results were as expected. 79 of the vaccinated volunteers had a significant neutralizing effect on their sera. In contrast, there was no significant neutralizing effect in the sera of the non-vaccinated volunteers, indicating that the method has great application value in neutralizing antibody monitoring. Furthermore, we compared this tri-mode method with other recently-reported methods for the detection of neutralizing antibodies (Table S3). It can be seen that, compared with neutralization test and ELISA, our method saved time to a large degree and reduced the requirement on biosafety level. While compred with other materials-based LFIA, our method supplied three detecton modes and achieved higher sensitivity. Thus, this tir-mode LFIA showed great application potential in practice.

Fig. 6.

Fig. 6

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Inhibition rate for the 107 serum samples (containing 9 non-vaccinated people and 98 vaccinated people) detected by the tri-mode LFIA platform.

4. Conclusions

In the present work, we created a tri-mode LFIA approach based on Au–Ag HNSs for qualitative SARS-CoV-2 neutralizing antibody detection with unaided eyes and quantitative analysis by photothermal and SERS spectroscopy. The Au-Ag HNSs possessed excellent photothermal effect, enhanced SERS signal, good stability, and achieved sensitive detection with a LOD of 20 ng/mL in serum samples, which was ten times lower that the visual observation. Moreover, the three kinds of signals (colorimetric, photothermal, and SERS signals) could complement one another, and the accuracy of the detection would be well improved. Additionally, the method had the advantages of simple operation, time efficiency, high specificity, and good reproducibility. The method was ultimately found to be successful in analyzing serum samples. As a result, particularly in areas with limited resources, our tri-mode LFIA technology shown considerable promise for neutralizing antibody surveillance. In the future, we plan to work in two directions. We will attempt to fix the probe on the conjugate pad to develop dry LFIA. Additionally, in order to speed up the testing procedure, we will attempt to directly detect whole blood samples rather than serum samples. The operation will become more practical and user-friendly as a result of these upgrades.

CRediT authorship contribution statement

Tianyu Zhao: Conceptualization, Methodology, Investigation, Writing – original draft. Penghui Liang: Investigation, Visualization, Writing – original draft. Jiaqi Ren: Investigation. Jinyue Zhu: Investigation. Xianning Yang: Investigation. Hongyu Bian: Investigation. Jingwen Li: Investigation. Xiaofeng Cui: Investigation. Chunhui Fu: Investigation. Jinyan Xing: Conceptualization, Methodology, Writing – review & editing. Congying Wen: Conceptualization, Methodology, Supervision, Writing – review & editing. Jingbin Zeng: Conceptualization, Methodology, Supervision, Writing – review & editing.

Declaration of competing interest

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.

Acknowledgments

This work was supported by the Key Fundamental Project of Shandong Natural Science Foundation (Z.J., ZR2020ZD13), the Natural Scientific Foundation of Shandong (W.C., ZR2020MB064, Z.J., ZR2022JQ07), the Fundamental Research Funds for the Central Universities (L.J., 21CX06014A; W.C., 22CX03033A), the Science and Technology Projects of Qingdao (Z.J., 21-1-4-sf-7-nsh), the National Natural Science Foundation of China (Z.J., no. 21876206), and the Taishan Scholarship of Shandong Province (tsqn202211080).

Handling Editor: Prof Rebecca Lai

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2023.341102.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.docx (327KB, docx)

Data availability

No data was used for the research described in the article.

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