Title： An integrated colorimetric and photothermal lateral flow immunoassay based on bimetallic Ag–Au urchin-like hollow structures for the sensitivedetection of E. coli O157:H7

Journal：Biosensors & Bioelectronics

IF：12.6

Original link：https://doi.org/10.1016/j.bios.2023.115090

Reporter：Xiaoling Zhang, Master of Grade 2022

Lateral flow immunoassay (LFIA) with gold nanoparticles (AuNPs) as the signal reporter is widely used for therapid detection of food-borne pathogens. However, it is difficult for LFIA to achieve sensitive detection due to theinsufficient colorimetric signal brightness of AuNPs. Herein, we developed a bimetallic Ag–Au urchin-like hollow nanospheres (BUHNPs) based on the simple and rapid synthesis through co-reduction and galvanic replacement reactions. The BUHNPs exhibit superb colorimetric signal brightness, strong photothermal signals and high antibody coupling efficiency.

1.Colorimetric LFIA (CM-LFIA) has the advantages of simplicity, portability, rapidity, and affordability.However, LFIA based on gold nanoparticles (AuNPs), i.e., one of most used nanomaterials, failed to achieve sensitive detection because of the lack of colorimetric signal brightness. There is an urgent need to develop colorimetric LFIA probes with easy synthesis, excellent colorimetric signals and favorable biocompatibility for the sensitive detection.

2.New alloy nanoparticles (NANPs) have attracted significant attention due to their unique optical and photothermal properties, which can be captured by handheld infrared cameras to improve the sensitivity of LFIA.

1、 Synthesis and characterization of BUHNPs

The costructure of the core was prepared by co-reduction and electrochemical replacement reaction using seed mediated growth method, which can be used as a promising photothermal material.

Fig. 1. Synthesis and characterization of BHNCs and BUHNPs. (A) Schematic illustration of the synthetic process of BHNCs and BUHNPs. (B) TEM images of BHNCs (The inset showed a modeling diagram of the nanoparticles). (C) SEM images of BHNCs. (D) Corresponding dark-field images scanning and EDS elemental mapping ofAu, Ag from BHNCs. (E) Hydrodynamic diameter of BHNCs. (F) TEM images of BUHNPs (The inset showed a modeling diagram of the nanoparticles). (G) SEM imagesof BUHNPs. (H) Corresponding dark-field images scanning and EDS elemental mapping of Au, Ag from BUHNPs. (I) Hydrodynamic diameter of BUHNPs. (J) XPS spectrum of BUHNPs. (K) Photographs taken from aqueous suspensions of AgNPs, AuNPs, BHNCs and BUHNPs. (L) Corresponding absorption spectra recorded from the samples. (M) The integrated area of visible light (400–800 nm) absorption of AgNPs, AuNPs, BHNCs and BUHNPs.

2、Research of photothermal properties of BUHNPs                                         

After exploring the composition and structure of the material, the photothermal properties of BUHNPs were further investigated.

Fig. 2. Photothermal performance characterization of BUHNPs. (A) Photothermal images of ultrapure water, AgNPs, AuNPs, BHCNs and BUHNPs upon irradiation of an 808 nm laser. (B) Photothermal heating curves of ultrapure water, AgNPs, AuNPs, BHCNs and BUHNPs under NIR light irradiation for 10 min (808 nm, 1.5 W cm−2). (C) Heating curves of 0.5 mL of aqueous solutions containing different amount of BUHNPs over time. (D) Heating curves of 0.5 mL of BUHNPs (100 μg mL−1) aqueous solutions upon irradiation of an 808 nm laser at different power densities. (E) Photothermal stability of BUHNPs upon four cycles of the on/off NIR laser. (F)Linear fitting plot of ln(θ)-time during the cooling period. (G) Schematic illustration of the multiple reflection effect.

3、Comparison of antibody coupling efficiency between AuNP and BUHNP

After exploring the photothermal properties, the high affinity of BUHNP surface was verified.

Fig. 3. Evaluation of the coupling efficiency of BUHNPs-antibody and AuNPs-antibody. (A) Schematic illustration of the process of coupling antibodies to BUHNPs and AuNPs. (B) Regression equation of OD450 nm versus anti-E. coli O157:H7 mAb concentrations. (C) Coupling efficiency of BUHNPs under various antibody concentrations. (D) Coupling efficiency of AuNPs under various antibody concentrations. (E) Colorimetric signal of BUHNPs-LFIA and AuNPs-LFIA at different concentrations of anti-E. coli O157:H7 mAb. (F) Photograph taken from BUHNPs-LFIA and AuNPs-LFIA at different concentrations of anti-E. coli O157:H7 mAb. (G)The effect of AA buffer solution on the coupling efficiency of nanoparticles with mAb. The concentrations of E. coli O157:H7 and mAb were 1 × 105 CFU mL−1 and 10μg mL−1. The standard deviation was calculated from the results of three independent tests.

4. Exploration of analytical performance of BUHNPs-LFIA

It is concluded that LFIA based on BUHNPs has high sensitivity, good specificity and can be used for actual sample detection.

Fig. 4. Analytical performance of BUHNPs-LFIA for E. coli O157:H7 detection. (A) The reacted test strip prototype photographs of BUHNPs-CM-LFIA (i), AuNPs-LFIA (ii), and BUHNPs-PT-LFIA (iii) in the qualitative detection of various E. coli O157:H7 concentrations ranged from 0 to 5 × 106 CFU mL−1. (B) The signal intensity of T line for E. coli O157:H7 quantitative detection based on AuNPs-LFIA, BUHNPs-CM-LFIA and BUHNPs-PT-LFIA. (C) Illustration of the linear dynamic range for quantitative detection of E. coli O157:H7 (Yellow line: BUHNPs-PT-LFIA; Blue line: BUHNPs-CM-LFIA; Red line: AuNPs-LFIA). (D) The LOD of AuNPs-LFIA, BUHNPs-CM-LFIA and BUHNPs-PT-LFIA in the assay of E. coli O157:H7. (E) The specificity of BUHNPs-LFIA was assessed by monitoring the signal changes in T line after responding to other interfering bacteria (Inset: the temperature changes of the T line base on BUHNPs-PT-LFIA). (F) A heat map of the recovery rates of food samples and municipal water based on the proposed BUHNPs-CM-LFIA and BUHNPs-PT-LFIA.

In summary, we propose dual-readout BUHNPs synthesized by co-reduction and current displacement and demonstrate their application as powerful signal probes to improve the detection of E.coli O157: H7. The obtained BUHNP showed excellent optical properties, photothermal properties and high antibody coupling efficiency, demonstrating the potential of this novel alloy nanomaterial in improving the sensitivity of dual-read LFIA detection, and providing innovative ideas for the application of multi-functional probes in ultra-sensitive biosensor platforms.