The benefits of molecularly imprinted polymer (MIP) technology in creating artificial receptors to replace natural counterparts have piqued the interest of numerous researchers in recent years. We propose a biomimetic enrofloxacin-MIP for enrofloxacin (ENRO) antigen detection using gold nanoparticles (AuNPs) and MIP methodologies in this study. A self-assembled monomer layer of aminothiophenol was used to immobilize a pre-formed complex of the anti-enrofloxacin monoclonal antibody and enrofloxacin antigen onto the surface of an AuNP coated screen-printed carbon ink electrode (SPCE). The poly-(aminothiophenol) layer thickness was adjusted to entrap and restrict enrofloxacin antigens near the surface. The imprinting and removal of the enrofloxacin antigen in the MIP film were strongly validated by the Raman spectra. The final mAb-MIP sensor had better sensitivity (302 Ω mL ng⁻¹) and a better detection limit (0.05 ng mL⁻¹) than self-assembled monolayer (SAM)-based immunosensors, which had 102 Ω mL ng⁻¹ and 0.1 ng mL⁻¹, respectively.

Klebsiella pneumoniae, a member of the family Enterobacteriaceae, is a rod-shaped, Gram-negative bacterium, mainly found in the hospital environment and medical tools. It is the leading cause of nosocomial infection, characterized by bloodstream infection, wound site infection, urinary tract infection, and sepsis, mostly in older adults, newborn infants, and immunocompromised patients. This present study demonstrated a novel diagnostic method for K. pneumoniae detection based on the gold nanozyme activity for the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H₂O₂. The nanozyme activity of AuNPs with staining enhancement was statistically three times higher than that of the bare AuNPs in solid absorption at 650 nm. Nano-ELISA with staining enhancement could detect as low as 10² CFUs/mL of K. pneumoniae concentration, as the cutoff value was determined to be 0.158, which boosted the sensitivity of the immunoreactions by up to 100-fold. The detection limit of our assays was 26.023 CFUs/mL, and the limit of quantification was 78.857 CFUs/mL. There was no cross-reaction against other bacteria, which proved the immunoassays' remarkable specificity for recognizing K. pneumoniae. Taken together, we successfully developed and optimized the highly sensitive and decently specific nano-ELISA strategy that might be applicable for detecting various other bacterial pathogens.

Lipopolysaccharides (LPS), the major components of Gram-negative bacterial outer membranes, are critical virulence factors that can trigger severe immune responses, leading to sepsis and other inflammatory diseases. Rapid and accurate LPS detection is therefore essential in clinical diagnosis, pharmaceutical safety, and food monitoring. Aptamers represent a promising alternative to antibodies due to their high specificity, stability, and cost-effectiveness. However, the conventional SELEX process for aptamer selection is labor-intensive and time-consuming, often generating numerous candidates requiring costly experimental validation. Herein, we present a computationally assisted post-SELEX refinement pipeline that integrates molecular docking and molecular dynamics simulations to efficiently prioritize and validate aptamer candidates obtained from SELEX. Using LPS from Klebsiella pneumoniae ATCC 15380 as the model target, ten rounds of SELEX yielded candidate ssDNA sequences that were subsequently evaluated through our computational workflow. Among the refined candidates, aptamer seq. 5 exhibited the highest predicted and experimental affinity, with a dissociation constant (K_d) of 6.68 nM, supported by both in vitro assays and molecular simulations. These finding demonstrate that the proposed SELEX-simulation pipeline significantly accelerates post-SELEX aptamer refinement, effectively reducing the number of candidates requiring expensive experimental screening and validation.

Pseudomonas aeruginosa is a clinically significant pathogen with high antibiotic resistance, necessitating rapid and reliable diagnostic methods. In this study, we developed a whole-cell aptamer selection method for P. aeruginosa using an Eppendorf-tube-based SELEX system, where bacterial cells were directly incubated with an ssDNA library. This configuration enhanced the recovery of bound aptamers and overcame the cell quantity limitations often encountered in microtiter-plate-based SELEX. After 10 selection rounds, six aptamer candidates were obtained and evaluated for affinity. Molecular docking analysis revealed that aptamer T1 possessed the highest target selectivity. To demonstrate diagnostic applicability, aptamer T1 was integrated into a hybrid lateral flow immunoassay (LFIA), replacing the conventional detection antibody. In this format, the AuNP–aptamer complex bound to the target bacteria and was captured by a specific antibody immobilized on the test line. The LFIA achieved a visual detection limit of 2.34 × 10² CFU/mL within 15 min, showing high specificity and suitability for point-of-care applications. This study presents the first demonstration of an aptamer–antibody hybrid LFIA for bacterial detection and highlights the potential of aptamers as low-cost, rapidly synthesized recognition elements adaptable for the detection of other infectious agents.

This study presents the design and application of an electrochemical sensor for selective detection of lead ions (Pb²⁺) based on ionophore-modified raspberry-like Fe₃O₄–Au nanostructures. The material was engineered with a magnetic Fe₃O₄ core, coated with polyethyleneimine (PEI) to facilitate nucleation, and subsequently decorated with Au nanoparticles, providing a raspberry-like (Fe₃O₄@PEI@AuNPs) nanostructure with high surface area and excellent electrochemical conductivity. Surface functionalization with Lead Ionophore IV (ionophore thiol) introduced Pb²⁺-selective binding sites, whose presence and structural evolution were verified by TEM and Raman spectroscopy. The Fe₃O₄ core endowed strong magnetic properties, enabling facile manipulation and immobilization onto screen-printed carbon electrodes (SPCEs) via physical adsorption, while the Au nanoparticles enhanced electron transfer, supplied thiol-binding sites for stable ionophore anchoring, and increased the effective electroactive surface area. Operational conditions were systematically optimized, with acetate buffer (HAc/NaAc, pH 5.7) and chronoamperometric preconcentration (CA) at −1.0 V for 175 s identified as optimal for differential pulse voltammetry (DPV) measurements. Under these conditions, the sensor exhibited a linear response toward Pb²⁺ from 0.025 mM to 2.00 mM with superior sensitivity and reproducibility compared to conventional AuNP-modified SPCEs. Furthermore, the ionophore-modified Fe₃O₄–Au nanostructure-based sensor demonstrated outstanding selectivity for Pb²⁺ over competing heavy metal ions (Cd²⁺, Hg²⁺, Cr³⁺), owing to the specific coordination interaction of Lead Ionophore IV with target ions. These findings highlight the potential of raspberry-like Fe₃O₄@PEI@AuNP nanostructures as a robust and efficient electrochemical platform for the sensitive and selective detection of toxic heavy metal ions.