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A new flow-through method for rapid capture and detection of microorganisms is developed using optically-flat microengineered membranes. Selective and efficient capture of Salmonella is demonstrated with antibodies coated on membranes (microsieves) having a pore size much larger than the microorganism itself. The silicon-nitride membranes are first photochemically coated with 1,2-epoxy-9-decene yielding stable Si–C and N–C linkages. The resultant epoxide-terminated microsieves are subsequently biofunctionalized with anti-Salmonella antibodies. The capture efficiency of antibody-coated microsieves with different pore sizes (2.0–5.0 μm) is studied with Salmonella enterica enterica serotype Typhimurium suspensions (107 cfu mL–1). The antibody-coated microsieves capture 52% (2 μm microsieves), 30% (3.5 μm microsieves), and 12% (5 μm microsieves) of Salmonella from the suspension. The influence of flow rate (0.8–16 μL min–1 mm–2) on the capture efficiency of antibody-coated 3.5 μm microsieves is investigated. The capture efficiency increases from ≈30% to ≈70% when the flow-rate decreases from 16 to 0.8 μL min–1 mm–2. Antibody-coated 3.5 μm microsieves can capture Salmonella rapidly and directly from fresh milk suspension (capture 35% at concentration of 80 cfu mL–1). The use of antibody-coated microsieves as microbial selective capture devices is thus shown to be highly promising for the direct capture of microorganisms.

Background Diagnostics and disease-management strategies require technologies to enable the simultaneous detection and quantification of a wide range of pathogenic microorganisms. Most multiplex, quantitative detection methods available suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. Here, we demonstrate the efficacy of a novel, high-throughput, ligation-based assay for simultaneous quantitative detection of multiple plant pathogens. The ligation probes, designated Plant Research International-lock probes (PRI-lock probes), are long oligonucleotides with target complementary regions at their 5' and 3' ends. Upon perfect target hybridization, the PRI-lock probes are circularized via enzymatic ligation, subsequently serving as template for individual, standardized amplification via unique probe-specific primers. Adaptation to OpenArrays™, which can accommodate up to 3072 33 nl PCR amplifications, allowed high-throughput real-time quantification. The assay combines the multiplex capabilities and specificity of ligation reactions with high-throughput real-time PCR in the OpenArray™, resulting in a flexible, quantitative multiplex diagnostic system. Results The performance of the PRI-lock detection system was demonstrated using 13 probes targeting several significant plant pathogens at different taxonomic levels. All probes specifically detected their corresponding targets and provided perfect discrimination against non-target organisms with very similar ligation target sites. The nucleic acid targets could be reliably quantified over 5 orders of magnitude with a dynamic detection range of more than 104. Pathogen quantification was equally robust in single target versus mixed target assays. Conclusion This novel assay enables very specific, high-throughput, quantitative detection of multiple pathogens over a wide range of target concentrations and should be easily adaptable for versatile diagnostic purposes.