Development of an integrated method of concentration and immunodetection of bacteria

The microbial quality of water is a key aspect to avoid environmental and public health problems. The low pathogen concentration needed to produce a disease outbreak makes it essential to process large water volumes and use sensitive and specific methods such as immunoassays for its detection. In the present work, we describe the development of a device based on microfiltration membranes to integrate the concentration and the immunodetection of waterborne bacteria. A microfiltration membrane treatment protocol was designed to reduce the non-specific binding of antibodies, for which different blocking agents were tested. Thus, the proof of concept of the microbial detection system was also carried out using Escherichia coli as the bacterial pathogen model. E. coli suspensions were filtered through the membranes at 0.5 mL s−1, and the E. coli concentration measurements were made by absorbance, at 620 nm, of the resultant product of the enzymatic reaction among the horseradish peroxidase (HRP) bonded to the antibody, and the substrate 3,3′,5,5′-tetramethylbenzidine (TMB). The results showed that the homemade concentration system together with the developed membrane treatment protocol is able to detect E. coli cells with a limit of detection (LoD) of about 100 CFU in 100 mL.
The authors acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (BACSYS pro- ject CTQ2014-54553-C3-1-R). N.U. and O.C. acknowledge funding from the People Programme (Marie Curie Actions) of the 7th Framework Programme of the E uropean Union (FP7/2007-2013), TECNIO-spring program from the Agency for Business Competitiveness of the Government of Catalonia (ACCIÓ). J.J.E. ac- knowledges financial support from the Catalan Industrial Doctorate pro- gram and Waterologies. S.L. and N.P. acknowledge financial support from Gas Natural Fenosa.