Sensors and Actuators B 138 (2009) 532–538 Contents lists available at ScienceDirect Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb Parallel acoustic detection of biological warfare agents surrogates by means of piezoelectric immunochips Thomas Alava a , Nathalie Berthet-Duroure a , Cédric Ayela a , Emmanuelle Trévisiol a , Martine Pugnière b , Yannick Morel c , Pascal Rameil c , Liviu Nicu a,∗ a LAAS-CNRS, 7 avenue du Colonel Roche, 31077 Toulouse, France b CPBS-CNRS, Faculté de Pharmacie, 15 avenue Charles Flahault, 34093 Montpellier, France c Centre d’Etudes du Bouchet, DGA/DET/CEB, 5 rue Lavoisier, 91710 Vert le Petit, France article info Article history: Received 15 December 2008 Received in revised form 9 February 2009 Accepted 20 February 2009 Available online 17 March 2009 Keywords: Biosensors Bioassays Quartz crystal microbalance Biological warfare agents abstract This paper focuses on flow functionalization of piezoelectric immunochips with antibodies against four different biological warfare agents (BWA) surrogates. To perform parallel detection of all BWA surro- gates at once, the E4 Quartz Crystal Microbalance with Dissipation monitoring system (QCM-D) is used. Assessment of antibodies immobilization, parallel detection of related BWA surrogates diluted in buffer solutions and regeneration of the complex antibodies/BWA surrogates are first discussed. Minimal detec- tion thresholds for Escherichia coli MRE 162, Bacillus atrophaeus, Cydia pomonella granulosis virus (CpGV) and ovalbumin are respectively equal to 2.4 × 10 7 CFU/mL, 1.4 × 10 6 spores/mL, 1.1 × 10 8 granules/mL and 1 g/mL. Detection experiments for three of the four BWA surrogates (E. coli MRE 162, B. atrophaeus and ovalbu- min) immersed in real liquid matrices from air sampler are successfully performed. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Intelligence sources have systematically warned of the risk of terrorist organizations attack using biological weapons such as anthrax, ricin, botulinum toxin, smallpox, plague or Ebola. To effi- ciently face this contemporary threat (namely for biodefense and risk evaluation), an early and definite identification of bacteria, viruses and toxins is of enormous importance. Real-time biosensors that can quickly, cheaply, and accurately detect airborne biological warfare agents (BWA) might be one of the solutions. Actually, these biosensors may be roughly divided into two classes: the first class relies on sort of “physical profiling” of the BWA of interest by means of mass spectrometry [1,2], modern infra-red [3,4] and Raman spec- troscopy [4,5], while the second class uses a specific biological recognition step that is provided by an appropriate ligand-receptor binding, such as antibody/antigen binding [6,7] or complementary binding of specific oligonucleotides to target DNAs [8]. In order to provide real-time detection and identification of BWA, the phys- ical profiling methods target “marker” molecules specific only to the agent(s) to be detected through complete biological sample Abbreviations: BWA, biological warfare agents; QCM, quartz crystal microbal- ance; SAM, self-assembled monolayer; MUA, mercaptoundecanoic acid; PBS, phosphate-buffered saline. ∗ Corresponding author. Tel.: +33 5 61 33 78 38; fax: +33 5 61 55 35 77. E-mail address: nicu@laas.fr (L. Nicu). preparation/analysis (including collection, concentration, lysis, and analysis of the sample [9]). In case of biological recognition-based systems, the event of recognition/identification is revealed and reported in a certain measurable way. Antibody/antigen binding recognition-based systems (also called “immunosensors”) generally rely on highly sensitive devices (or transducers) to translate the biological recognition event into a physical signal variation. More precisely, antibodies are “grafted” onto the active surface of the transducer so that the event of antigen binding triggers changes of the transducer’s physical surface state (thus inducing readout signal variation) such as refractive indices of the layer in Surface Plasmonic Resonance case [10–12], orientation of molecules within the layer in “liquid-crystal” transducers [13] or the layer’s weight/thickness in Quartz Crystal Microbalance (QCM) transducer [14,15]. Compared to the other antibody/antigen binding recognition techniques that often suffer from either long analysis time, compli- cated procedures, non-portability or high costs [16], acoustic-based sensors (such as QCM) have attracted considerable interest for the development of BWA sensors. The QCM exploits the piezoelectric properties of a quartz crystal disc such as when an electric field is applied across electrodes placed on both sides of the crystal, it leads to a physical deformation of the disc (due to the so-called inverse piezoelectric effect) [17]. Perturbation of the resonant frequencies of the crystal is attributed to a change of mass on the modified electrode surface. The frequency and mass change on a QCM crys- tal surface is expressed by the well-known Sauerbrey equation 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.02.060