title: Automated microfluidically controlled electrochemical biosensor for the rapid and highly sensitive detection of Francisella tularensis.
abstract: Tularemia is a highly infectious zoonotic disease caused by a Gram-negative coccoid rod bacterium, Francisella tularensis. Tularemia is considered as a life-threatening potential biological warfare agent due to its high virulence, transmission, mortality and simplicity of cultivation. In the work reported here, different electrochemical immunosensor formats for the detection of whole F. tularensis bacteria were developed and their performance compared. An anti-Francisella antibody (FB11) was used for the detection that recognises the lipopolysaccharide found in the outer membrane of the bacteria. In the first approach, gold-supported self-assembled monolayers of a carboxyl terminated bipodal alkanethiol were used to covalently cross-link with the FB11 antibody. In an alternative second approach F(ab) fragments of the FB11 antibody were generated and directly chemisorbed onto the gold electrode surface. The second approach resulted in an increased capture efficiency and higher sensitivity. Detection limits of 4.5 ng/mL for the lipopolysaccharide antigen and 31 bacteria/mL for the F. tularensis bacteria were achieved. Having demonstrated the functionality of the immunosensor, an electrode array was functionalised with the antibody fragment and integrated with microfluidics and housed in a tester set-up that facilitated complete automation of the assay. The only end-user intervention is sample addition, requiring less than one-minute hands-on time. The use of the automated microfluidic set-up not only required much lower reagent volumes but also the required incubation time was considerably reduced and a notable increase of 3-fold in assay sensitivity was achieved with a total assay time from sample addition to read-out of less than 20 min.
authors.affiliation: Nanobiotechnology and Bioanalysis Group, INTERFIBIO, Departament d׳Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain. Electronic address: samuelbacena.dulay@estudiants.urv.cat.
authors.forename: Samuel B
authors.initials: SB
authors.lastname: Dulay
authors.affiliation: Fraunhofer ICT-IMM, Carl-Zeiss-Strasse 18-20, 55129 Mainz, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Gransee
authors.affiliation: Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut, Naumburger Strasse 96 a, Jena 07743, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Julich
authors.affiliation: Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut, Naumburger Strasse 96 a, Jena 07743, Germany.
authors.forename: Herbert
authors.initials: H
authors.lastname: Tomaso
authors.affiliation: Nanobiotechnology and Bioanalysis Group, INTERFIBIO, Departament d׳Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain; Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys, 23, 08010 Barcelona, Spain. Electronic address: ciara.osullivan@urv.cat.
authors.forename: Ciara K
authors.initials: CK
authors.lastname: O'Sullivan
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation*; D055664:Electrochemical Techniques / Q000295:instrumentation; D004867:Equipment Design; D005604:Francisella tularensis / Q000302:isolation & purification*; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation; D056656:Lab-On-A-Chip Devices; D008070:Lipopolysaccharides / Q000032:analysis*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008958:Models, Molecular; D014406:Tularemia / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D055910:Antibodies, Immobilized; D008070:Lipopolysaccharides
keywords: Electrochemical immunosensors; Francisella tularensis; Lab-on-a-chip; Self-assembled monolayer (SAM)