A reinforced composite structure composed of polydiacetylene assemblies deposited on polystyrene microspheres and its application to H5N1 virus detection

Wenjie Dong,¹ Jing Luo,² Hongxuan He,² Long Jiang<sup>1

1</sup>Beijing National Laboratory for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing, People's Republic of China; ²National Research Center for Wildlife Born Diseases, Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China

Abstract: In this study, we immobilized polydiacetylene vesicles (PDAVs) onto the surface of polystyrene microspheres (1 µm in diameter) by using both electrical charge and conjugated forces to form a reinforced composite structure. These reinforced complexes could be easily washed, separated by centrifugation, and resuspended by gentle agitation. After passing through a narrow 200 µm-diameter channel, the composite structures maintained their original shape, demonstrating their resilience and potential for use in microfluidic technologies. The number of PDAVs in the composite structure could be mediated by changing the extent of layer deposition, which affected the sensitivity of detection. It showed that PDAVs did not change their blue color after addition of detecting probes such as anti-H5N1, which was of great importance in the fabrication and modification of stable color-changeable biosensors based on PDAVs. By conjugating anti-H5N1 antibodies to the PS@PDAV via N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide chemistry, a stable blue complex, anti-H5N1 microsphere (PS@PDAV-anti-H5N1) was formed. A target antigen of H5N1 (HAQ [H5N1 strain A/environment/Qinghai/1/2008{H5N1} in clade 0]) was detected by PS@PDAV-anti-H5N1. At an optimal PDAV deposition level of three layers, the limit of detection was determined to be approximately 30 ng/mL of HAQ by using optical spectrum measurement and visual inspection, meeting the needs of fast and simple color-changeable detection. However, a much lower limitation of detection (1 ng/mL) was able to be obtained using laser-scanning confocal microscopy, which could be compared with the results obtained with other sophisticated equipment.

Keywords: reinforced composite structure, polystyrene, polydiacetylene, H5N1 virus detection