(I-359) Gold Nanostar-Based Vertical Flow Immunoassay for Detection of Biothreat Agents

Introduction:: The paper-based vertical flow immunoassay (VFI) has a demonstrated ability for the detection of multiple biothreat agents, including high priority Tier 1 agents such as Melioidosis, Anthrax, Plague and Tularemia with high sensitivity and multiplex capability [1,2]. VFI configuration has enabled enhanced limit of detection for these select agents compared to their lateral flow immunoassay (LFI) counterparts [3,4]. Recently, sharp branched gold nanostars have been used to enhance colorimetric detection of Y. pestis in LFIs [5]. This study sought to evaluate the use of gold nanostars for the colorimetric detection of Y. pestis in VFI compared to the use of traditional gold nanospheres.

Materials and Methods:: For this study, VFI membranes were prepared using a Nano-plotter 2.1 to dispense capture antibody reagents onto a nitrocellulose substrate in a clean room environment. Samples were prepared by spiking recombinant protein into buffer and incubating with ~ 250 nm gold nanostar-labeled detection antibodies for 10 min. The VFI membranes were then assembled into custom housings, and pre-incubated samples were filtered (0.2 µm PES) before being loaded into 5 mL luer-lock syringes and passed through the membrane using a syringe pump at 0.2 mL/min. After passing the samples, membranes were dried and scanned for image analysis.

Results, Conclusions, and Discussions:: In this study, gold nanostar-labeled detection antibodies were used for the first time to detect the F1 antigen of Y. pestis in VFI. Based on signal intensities from the performed assays, the target antigen could be detected as low as 31.25 pg/mL. This improved sensitivity can be attributed to the branches of this anisotropic gold nanoparticle system. Nanostars have the advantages of increased surface area and strong localized surface plasmon resonance (SPR) absorption. Increasing surface area carries the advantage of higher loading capacity for detection antibodies. Whereas the strong SPR absorbance improves light scattering properties and yields strong optic absorption with very large molar extinction coefficients. Together, these qualities of gold nanostars demonstrate that they can provide enhanced detection of target antigens for immunoassays, such as VFI and LFI, compared to traditional gold nanospheres.

VFIs have proven the ability to increase immunoassay sensitivity compared to standard LFIs using traditional gold nanospheres. Gold nanostars have shown the ability to add additional enhancement of sensitivity due to their advantages with surface area and optical properties. Together, VFI with gold nanostars could be used as an analytical tool to enhance detection of targets with low LFI sensitivity.


Acknowledgements (Optional): : This work was supported by the Chemical Biological Technologies Directorate (Contract # HDTRA1-16-C-0026) and the Advanced Technology International (Contract # MCDC-18-04-09-002) from the Department of Defense Chemical and Biological Defense program through the Defense Threat Reduction Agency (DTRA).

References (Optional): : 1. Chen, Peng et al. Talanta vol. 191, 0039-9140. 1 Jan. 2019

2. Devadhasan, Jasmine et al. Biosensors and Bioelectronics vol. 219, 0956-5663. 1 Jan. 2023

3. Hau, Derrick et al. PLOS Neglected Tropical Diseases vol. 16, 3, e0010287. 23 Mar. 2023

4. Hannah, Emily et al. Pathogens vol. 10, 8, 924. 22 July 2021

5. Atta et al., ACS Appl. Nano Mater. 2023, 6, 5, 3884–3892