Electrochemical aptasensor for trimethoprim detection based on Pd@Ir bimetallic nanoparticles coupled with hybrid chain reaction

Trimethoprim (TMP), a widely used veterinary antibacterial, accumulates in animal-derived foods and poses health risks like bacterial resistance and bone marrow hematopoietic suppression. Herein, an ultrasensitive electrochemical aptasensor was developed by integrating molecular docking-optimized TMP-specific aptamers, Pd@Ir bimetallic nanoparticles (Pd@Ir NPs) for signal enhancement, and hybrid chain reaction (HCR) for signal amplification. First, a high-affinity TMP-specific aptamer TMP-1 was screened via Capture-SELEX. Through molecular docking to identify core binding sites and truncate redundant sequences, TMP-1 was optimized into the 56 nt TMP-1C with enhanced affinity (The Kd was determined to be 41.6 nM by SGI assays). Pd@Ir NPs with excellent conductivity and electrocatalytic activity were coupled to hairpin H2 to form Pd@Ir NPs@H2 probes. For sensor construction, thiol-modified cDNA-TMP-1C duplexes were immobilized on gold electrodes, the binding between TMP and aptamer induced the duplex de-hybridization, and the exposed cDNA opened H1 and triggered HCR, thereby achieving Pd@Ir NPs enrichment and signal amplification. This method exhibits an ultra-low limit of detection (LOD) of 0.02 ng/mL. The spiked recovery experiments on pork samples show that the recovery rate of TMP ranges from 92.00 % to 93.99 % with a relative standard deviation (RSD) of 3.34 %–3.89 %, confirming its accuracy in practical samples. This sensor provides an efficient, high-performance tool for TMP residue detection in food safety monitoring.