DNA Hydrogel-Based Fluorescence Biosensor Packed in a Three-Dimensional-Printed Microextractor for Direct Tracking of...

The free antibiotic resistance genes (ARGs) content in water samples both inside (during treatment) and outside (after treatment) a wastewater treatment plant (WTP) is of great significance for evaluating the effect of treatment and environmental risks. In this work, a functional DNA hydrogel was packed in a three-dimensionally (3D)-printed tube as a fluorescence biosensor-integrated microextractor to directly extract and track free ARGs from water environments and biological samples. Combined with the strand displacement amplification reaction and the 3D-printed tube in respect of its spatial confinement effect, the 1,1,2,2-tetra (4-carboxylbenzene) ethylene-based DNA hydrogel was used for rapid extraction and highly sensitive fluorescence detection of free ARGs. The limits of detection of free ARGs (bla_-TEM, tetM, sul-1 and bla-_CTX-M-1) were determined to be 0.165, 0.231, 0.171, and 0.159 fM, respectively, and the linear calibration ranges were from 1 fM to 100 nM. Furthermore, the proposed method was applied to analyze the distribution and transfer of free ARGs (represented by bla_-TEM) in water environments. The distribution of bla_-TEM at different sampling points (both inside and outside of the WTP) was tracked and analyzed. In addition, the dynamic changes of bla_-TEM in crucian carp after the introduction of the antibiotic penicillin G sodium into aquaculture water were investigated; horizontal transfer of bla_-TEM was also explored using Escherichia coli DH5α with introduced plasmids. This strategy provides a new solution for rapid, convenient, and accurate ARGs analysis in water samples and biological samples and has good applicability prospects in the environmental and bioanalytical fields.