An impedimetric sensor based on a three dimensional electrode array modified with concanavalin A (Con A) was used for label-free detection of bacterial endotoxin: lipopolysaccharide (LPS) from Escherichia coli. The transducer permits the detection of the surface charge changes due to interaction of immobilized Con A biorecognition element and LPS of E. coli in test solution. The deposition of Con A on the surface was carried out using the layer-by-layer method with polyethyleneimine (PEI) polycation as an initial layer. The sensor surface characterization by means of electrochemical impedance spectroscopy technique allowed registering variations in superficial resistance provoked by surface charge changes and is demonstrated as an effective method to monitor sensor parameters at each modification step as well as to follow Con A - LPS reaction. In order to prevent non-specific adsorption of LPS on PEI covered surface different blocking strategies were tested to achieve the specific response between Con A and LPS. Results obtained in this work clearly show that blocking with bovine serum albumin (BSA) is not sufficient to prevent non-specific interactions of PEI and to ensure the selective biorecognition of LPS by Con A. To achieve more efficient PEI blocking a new method was proposed based on consecutive deposition of Con A-glycogen-Con A layers. Sensors modified with PEI-(Con A-Gly)(2)-Con A multilayers are shown to be highly sensitive, selective and reproducible. Presented biosensor is able to detect bacterial LPS in a very short detection time (20 min) with 2 mu g mL(-1) limit of detection, which is much lower than reported for other biosensors with Con A. (C) 2017 Elsevier Ltd. All rights reserved.