Title： High-density Au nanoshells assembled onto Fe3O4 nanoclusters for integrated enrichment and photothermal/colorimetric dual-mode detection of SARS-CoV-2 nucleocapsid protein

Journals：Biosensors & Bioelectronics

Impact factor：12.6

Original link：https://doi.org/10.1016/j.bios.2023.115688

Reporter：Jiaqi Ma，Master of Grade 2022

This article developed a polyethylenimine (PEI)-mediated approach for assembling high-density Au nanoshells onto Fe3O4 nanoclusters (MagAushell) as LFIA labels for integrated enrichment and photothermal/colorimetric dual-mode detection of SARS-CoV-2 nucleocapsid protein (N protein). PEI layer served not only as “binders” to Fe3O4 nanoclusters and Au nanoshells, but also “barriers” to ambient environment. Thus, MagAushell not only combined magnetic and photothermal properties, but also showed good stability. With MagAushell, N protein was first separated and enriched from complex samples, and then loaded to the strip for detection. By observation of the color stripes, qualitative detection was performed with naked eye, and by measuring the temperature change under laser irradiation, quantification was attained free of sophisticated instruments. The introduction of Fe3O4 nanoclusters facilitated target purification and enrichment before LFIA, which greatly improved the anti-interference ability and increased the detection sensitivity by 2 orders compared with those without enrichment. Moreover, the high loading density of Au nanoshells on one Fe3O4 nanocluster enhanced the photothermal signal of the nanoprobe significantly, which could further increase the detection sensitivity. The photothermal detection limit reached 43.64 pg/mL which was 1000 times lower than colloidal gold strips.

1. Traditional colloidal gold-based LFIA usually has low sensitivity, cannot achieve accurate quantification and is susceptible to interference due the relatively low molar extinction coefficient of Au nanoparticles (NPs).

2. The photothermal conversion materials which can convert light into heat in the form of rising in temperature have shown great application advantages in the field of sensing due to the high signal-to-noise ratio of photothermal materials and high sensitivity of temperature readout device. Magnetic nanomaterials may be promising tools to solve this problem, due to the fact that magnetic materials can achieve rapid and low-cost separation and enrichment of target among a large number of specimens under an applied magnetic field. Therefore, nanomaterials combining photothermal and magnetic properties may achieve both high sensitivity and strong anti-interference ability.

1.

Scheme 1. Fabrication of MagAushell NPs probes and their application to LFIA-based biosensor for detection of SARS-CoV-2. (a) Flow chart of synthesis and functionalization of MagAushell NPs. (b) Schematic diagram of the structure and detection principle of the LFIA. (c) Interpretation of test results and the typical positive and negative results from visual and infrared thermal camera observation.

2.

Fig. 1. Characterization of Au nanoshells. (a) Photographs and UV–vis spectra of Au nanoshells synthetized using different amounts of HAuCl4. (Ag/Au molar ratio of 15:0, 15:1, 15:2, 15:3, 15:4, 15:5, 15:6, from 0 to 6). (b) TEM of Au nanoshells synthetized using Ag/Au molar ratio of 15:5. (c) Particle size distribution diagram of Au nanoshells synthetized using Ag/Au molar ratio of 15:5.

In order to obtain Au nanoshells whose surface plasmon resonance (SPR) peak matched the 808 nm laser to achieve high photothermal signal, authors investigated the influence of HAuCl4 amount on the SPR peak. By comprehensive consideration of the SPR peak and stability, Ag/Au molar ratio of 15:5 was selected. Under this condition, Au nanoshells with 760 nm SPR peak was achieved (Fig. 1a, red line), and the synthesized Au nanoshells appeared complete hollow polygon structure with average size of 31 nm (Fig. 1b–c).

3.

Fig. 2. Characterization of MagAushell NPs. (a–b) TEM images of MagAushell NPs. (c–f) EDX element mappings of Fe, O, Au, and the overlay. (g) Hysteresis loops of Fe3O4 nanoclusters and MagAushell NPs. (h) Heating and cooling curves of Au nanoshells, Fe3O4 nanoclusters, and MagAushell NPs using laser irradiation (808 nm, 1.44 W/cm2, 10 min). (i) Stability study of MagAushell NPs solution under the photothermal heating and cooling cycles using laser irradiation (808 nm, 1.1 W/cm2). (j) Zeta potential of Fe3O4 nanoclusters, Fe3O4 nanoclusters-PEI, Au nanoshells, MagAushell, and MagAushell-Ab NPs. (k) Photographs of the test strips loaded with MagAushell and MagAushell-Ab NPs.

The energy dispersive X-ray (EDX) element mapping of a MagAushell NP Fig.(2c-f) showed that the element of Fe and O distributed in the whole NP, and Au elements with hollow structure disturbed on the surface of Fe and O. Fig.2g indicated that MagAushell NPs had superparamagnetism. As Fig.2i MagAushell NP exhibited good photothermal stability.

4.

Fig. 3. (a) Images of test strips obtained from detection of different concentrations of N protein with magnetic enrichment. (b) Images of test strips obtained from detection of different concentrations of N protein without magnetic enrichment. (c) Calibration plot of photothermal signal versus N protein concentration and corresponding photothermal images. The black star presents the naked eye detection limit. Error bar = SD, n = 3.

As shown in Fig. 3a, when there was no N protein in the sample, the T-line did not show any colors and only the C-line produced a brown stripe. As the concentration of N protein increased, more and more MagAushell-Ab1-N protein accumulated on the T-line, the color of the T-line became darker and darker, and the brown stripe on the T-line was just visible when the concentration of N protein was increased to 1 ng/mL. Thus, the visual LOD of the LFIA was 1 ng/mL by observing with naked eye. As shown in Fig. 3b, its visual LOD was just 100 ng/mL without pre-enrichment. This meant that the sensitivity after enrichment was 100 times higher than that without enrichment, demonstrating the superiority of enrichment. Then the photothermal signals were measured using infrared light and infrared camera. As shown in Fig. 3c, the photothermal signal (ΔT) showed good linear relationship with N protein concentration from 100 pg/mL to 1000 ng/mL (R2 = 0.999). 

5.

Fig. 4. (a) Colorimetric images of test strips for detection SARS-CoV-2 N protein, Victoria, Yamagata, H1N1, and blank samples. (b) Histogram of photothermal signals obtained from specificity experiments and corresponding photothermal images. Error bar = SD, n = 3.

As shown in the figure 4, indicating that this LFIA had good reproducibility.

6.

Fig. 5. Colorimetric images of test strips for detection SARS-CoV-2 N protein in PBS and Saliva (a) and their photothermal data statistical graph (b). Error bar = SD, n = 3.

The N protein were added into artificial saliva to simulate real samples and the recovery experiments were performed. The above results indicated that this MagAushell-based LFIA had considerable potential and promise for practical detection of SARS-CoV-2 N protein.

In this paper, we proposed a PEI-mediated approach by assembling high-density Au nanoshells with high photothermal conversion efficiency onto the surface of Fe3O4 nanoclusters, and developed a dual-mode LFIA for detecting SARS-CoV-2 N protein. The photothermal quantitative LOD reached 43.64 pg/mL, which was 1000 times lower than that of colloidal gold strip.