The detection and recognition of analytes by molecular interactions has promoted recent efforts for the development and design of analytical protocols and devices both for biomedical and bioprocess applications. Traditionally transduction of biorecognition is achieved indirectly by gathering a signal generated by specific reporter groups, usually fluorescent or electrochemically active molecules, used to label the targeted analytes. Target labelling is a major bottleneck in such systems since it may be a source of error, irreproducibility, and contamination and is detrimental to the sensitivity of the overall system. To overcome such bottleneck new generation of biosensors are being developed aiming the direct transduction of the biorecognition event occurring at the surface of the transducer. Towards this objective, different physical transducers, such as surface plasmon resonance, piezoelectric and acoustic wave devices, are capable of measuring surface mass changes resulting from the formation of biocomplexes at a sensitive area.

Although mostly advanced optical systems are utilized, for example based on the surface plasmon resonance, piezoelectric devices generating bulk acoustic waves represent a similar but significantly less expensive alternative. In particular the popularity of piezoelectric devices has increased significantly as microgravimetric mass sensors in Quartz Crystal Microbalances (QCM) apparatus. QCM operation in liquid environments is however characterized by a sensitive response to the properties of solutions and adsorbed films, which leads to the misinterpretation of the measured data [1]. We have shown that these interferences can be isolated and quantified advanced impedance analysis, where the QCM is represented by the Butterworth–Van Dyke (BVD) model. The BVD model is and equivalent electrical circuit representation of the sensor composed by a static capacitance related with charge effects (C0) in parallel with a motional branch containing in series a motional inductance, related with mass adsorption (Lm), a motional capacitance, related with the sensor/crystal elasticity (Cm) and a motional resistance (Rm) which is related with viscoelastic effects. An advanced measurement system using a network (or impedance) analyzer enables the quantification of the different contributions of Rm, Lm, Cm and C0 for each stage of the biomolecular recognition process, by fitting the impedance magnitude (|Z|) complex equation to the measured spectrum around the resonance frequency of the crystal [1].

This communication focus the removal of such interfering signals in QCM based piezoelectric biosensors to measure bimolecular binding kinetics as well as to detect biomolecules in biological samples. The effect of charged species, density, viscosity and signal enhancement was investigated to optimize QCM performance in terms of binding monitoring [8]. The optimized methodology was further used to measure the binding kinetics of single chain (4BL) and single domain (VH and VHD) recombinant antibodies generated against HIV1-vif. The recombinant antibodies were immobilized onto activated sensors surface and used as biorecognition material. The antibody immobilization was followed by frequency counting and impedance analysis. Impedance analysis enabled the quantification and elimination of interfering signals from biofilm viscoelasticity or charge [1], thus enabling the analytical quantification of mass from resonance frequency variation data [2,3]. Antibodies adsorbed linearly to the sensor surface within the concentration range evaluated. All immobilized antibodies were able to specifically recognize HIV1 Vif in liquid samples [2]. No signal was obtained when complex protein mixtures were applied to the sensors surface while significant frequency variations were observed upon spiking with known amounts of purified HIV1-Vif protein [3]. It was found that the antibody modified sensors detected the target antigen differently. The combined frequency variation and impedance analysis data suggest that the different signals obtained during HIV1-Vif recognition by the antibody modified sensors is due to the antibody hydrophobicity which result in different antibody-to-surface orientation and eventual hindrance of recognition motives [3]. We further demonstrate the potential of these sensors as tools for HIV1 infection monitoring and follow-up through the successful selective detection of HIV1 vif from HEK293 cell culture extracts [3].

[1]Encarnação, JM, Stallinga, P, Ferreira, GNM, “Influence of electrolytes in the QCM response: Discrimination and quantification of the interference to correct microgravimetric data”, Biosens. Bioel. (2007) 22:1351-1358.

[2]Encarnação, JM; Rosa, L.; Rodrigues, R.; Pedro, L.; Aires da Silva, F.; Gonçalves, J.; Ferreira, GNM; Piezoelectric biosensors for biorecognition analysis: Application to the kinetic study of HIV-1 Vif protein binding to recombinant antibodies”, J. Biotechnol (2007) 132: 142-148.

[3]Ferreira, GNM; Encarnação, JM; Rosa, L.; Rodrigues, R.; Breyner, R.; Barrento, S.; Pedro, L.; Aires da Silva, F.; Gonçalves, J., “Recombinant single-chain and single domain antibody piezoimmunosensors for detection of HIV1 virion infectivity factor”, Biosens. Bioel. (2007) 23:384-392.

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