DNA Hydrogel-Based Fluorescence Biosensor Packed in a Three-Dimensional-Printed Microextractor for Direct Tracking of Free Antibiotic Resistance Genes in Wastewater Treatment Plants

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.