Preparation and mechanism study of hydrogen bond induced enhanced composited gelatin microsphere probe

Fluorescent probes offer rapid and efficient detection of metal ions. However, their properties, including high biotoxicity and low detection limits, often limit their utility in biological systems. In this study, we used a microfluidic approach to fabricate photocrosslinked gelatin microspheres with a micropore, providing a straightforward method for loading fluorescent probes into these microspheres based on the adsorption effect and hydrogen bonding interaction. The gelatin microsphere loaded probes, GelMA/TPA-DAP and GelMA/TPAISO-HNO were designed and obtained. The results show that these probes exhibit obviously low biotoxicity compared to the original molecular probes TPA -DAP and TPA-ISO-HNO. Simultaneously, it is found that GelMA/ TPA -DAP and GelMA/TPA-ISO-HNO have better detection sensitivity, the detection limits are 35.4 nM for Cu 2 + , 16.5 nM for Co 2 + and 20.5 nM for Ni 2 + for GelMA/TPA-DAP probe. Compared to the original TPA -DAP they are improved by 37.2 %, 26.3 % and 22.6 % respectively. The corresponding coordination constants were 10.8 x 10 5 , 4.11 x10 5 and 6.04 x10 5 , which is larger than homologous TPA -DAP. Similar results were also verified in the GelMA/TPA-ISO-HNO probe. The mechanism was investigated in detail by theoretical simulations and advanced spectral analysis. The density functional theory (DFT) simulations show that the probes are anchored inside the microspheres and the molecular structure is modified due to the hydrogen bonding interaction between the microsphere and the molecular probe, which makes GelMA/TPA-DAP exhibit stronger coordination capacity with metal ions than homologous TPA -DAP. In addition, the adsorption effect also provided some synergistic enhancement contribution. Meanwhile, cellular experiments have also shown that the composite microspheres can improve the biocompatibility of the probe and will provide a wider range of applications towards bioassay. This simple and effective method will provide a convenient way to improve the performance of fluorescent probes and their biological applications.