A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins Raquel B. Queirós, N. de-los-Santos-Álvarez, J.P. Noronha, M.G.F. Sales Keywords: DNA, Aptamer E. coli, OMPs, Biosensor, Faradaic impedance spectroscopy abstract A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane pro- teins (OMPs) was developed. Two single stranded DNA sequences were tested as recognition elements and compared. The aptamer capture probes were immobilized, with and without 6-mercapto-1-hexanol (MCH) on a gold electrode. Each step of the modification process was characterized by Faradaic impedance spectroscopy (FIS). A linear relationship between the electron-transfer resistance (R et ) and E. coli OMPs concentration was demonstrated in a dynamic detection range of 1 × 10 -7 –2 × 10 -6 M. Moreover, the aptasensor showed selectivity despite the presence of other possible water contaminates and could be regenerated under low pH condition. The developed biosensor shows great potential to be incorporated in a biochip and used for in situ detection of E. coli OMPs in water samples. 1. Introduction Escherichia coli (E. coli) is a bacterium that is commonly found in the gut of humans and other warm-blooded animals. While most strains are harmless, some of them can cause severe food- borne disease such as the E. coli O104:H4 outbreak in Germany in 2011. Infections caused by E. coli are usually transmitted through consumption of contaminated water or food, such as undercooked meat products and milk. The consumption of contaminated water can cause large and sometimes widely dispersed outbreaks. Symp- toms of disease include abdominal cramps, fever, vomiting and diarrhea, which may be bloody in the case of contamination by enterotoxic E. coli (ETEC) or enterohemorragic E. coli (EHEC). Most patients recover within 10 days, although in a few cases the disease might become life-threatening [1,2]. The routine detection meth- ods for these microorganisms are based on colony forming units (CFU) counting requiring selective culture, and biochemical and serological characterizations. Regardless these methods are sen- sitive and selective, they need a lot of time to get a result. Besides, these methods are costly and time consuming [3]. The rapid evo- lution of the symptoms requires a rapid, sensitive and reliable monitoring of bacterial contamination. Biosensors can assume this role in this case [4,5], which in combination with electrochemical techniques for transduction of protein recognition can provide the simplicity and speed required. Antibodies are the molecular recognition element commonly chosen but they present some limitations such as limited shelf life, thermal and chemical instability leading to denaturation of proteins and loss of binding ability, and target restriction to immunogenic molecules that do not represent constituents of the body [5,6]. As an alternative, aptamers (APTs) are oligonucleotides, DNA or RNA molecules, which can interact with high specificity and affinity to their targets [7,8], due to its ability to fold into numerous tertiary conformations. Aptamers can be generated by a combinatorial procedure called “systematic evolution of ligands by exponential enrichment” (SELEX) [9] and synthesized in a large quantity in vitro in a very reproducible way [10,11]. Several aptamers have been developed to bind different targets, like proteins, small molecules, cells, antibi- otics, and viruses [10], in various types of biodetection approaches from label-free [12] to self-reporting or labeling strategies [13] using a variety of assay formats [14]. Evolution of aptamers against bacteria is an underexplored area. Unlike conventional SELEX, the specific target is not usually known and a pool of aptamers against different targets is often found. This obliges to carry out further experiments in order to know the exact nature of the final target if desired. To avoid this cumbersome step,