Piezoelectric biosensors assisted with electroacoustic impedance spectroscopy: a tool for accurate quantitative molecular recognition analysis.

In this work, electroacoustic impedance analysis based on a modified Butterworth-Van Dyke (BVD) model is used to complement resonance frequency measurements of piezoelectric crystal sensors for the identification and removal of interfering signals. This approach enables the accurate use of the Sauerbrey correlation to establish a direct relationship between mass deposited at the sensor surface and measured frequency variations. Kinetic models can thus be evaluated and binding constants estimated directly from the measured data. We further demonstrate the usefulness of this approach by applying it to the study of the formation of 11-hydroxy-1-undecanothiol self-assembled monolayers (SAM) as well as to the binding of streptavidin to immobilized biotin. Kinetic and equilibrium parameters were estimated from transient analysis, adsorption isotherms, Scatchard and Hill plots obtained from the frequency data for both the alkanethiol and streptavidin films. This strategy based on electroacoustic impedance assisted quartz-crystal microbalance (QCM) biosensors is expected to be a major contribution for the use of these piezoelectric devices as a reliable and cheap detection system that can easily be integrated into analytical techniques.

Piezoelectric biosensors for biorecognition analysis: application to the kinetic study of HIV-1 Vif protein binding to recombinant antibodies.

In this work three piezoelectric sensors modified with anti-HIV-1 Vif (virion infectivity factor) single fragment antibodies (4BL scFV), single domains (VH) and camelized single domains (VHD) were constructed and used to detect HIV1 Vif in liquid samples. Dithio-bis-succinimidyl-undecanoate (DSU) and 11-hydroxy-1-undecanethiol (HUT) mixed self assembled monolayers (SAM) were generated at the sensors surface onto which the antibodies were immobilized. All sensors detected specifically the target HIV1-Vif antigen in solution and no unspecific binding was monitored. Impedance analysis was performed to quantify electroacoustic and viscoelastic interferences during antibody immobilization and antigen recognition. The elimination of such interferences enabled the quantitative use of the piezoelectric immunosensors to estimate the antibody surface density as well as antigen binding and equilibrium constants. In spite of the possible limitation regarding mass transport and other related molecular phenomena, which were not considered in the binding model used, this work demonstrates the usefulness of piezoelectric biosensors in biorecognition analysis and evidences the advantages on using simultaneous impedance analysis to bring analytical significance to measured data, and thus to improve piezoelectric sensors sensitivity and applicability.

Recombinant single-chain variable fragment and single domain antibody piezoimmunosensors for detection of HIV1 virion infectivity factor.

In this paper recombinant single-chain fragments (scFv-4BL), and single domain antibodies (4BL-V(H)) and (4BL-V(H)D) generated against HIV1 virion infectivity factor (Vif) are used to develop piezoimmunosensors for HIV1 recognition. Mixed self assembled monolayers were generated at the surface of gold coated crystal sensors to which scFv-4BL, 4BL-V(H), or 4BL-V(H)D were immobilized. Impedance analysis was used to discriminate interfering signals from frequency variation data and to increase the sensor sensitivity. The elimination of interfering signals enabled the quantification of the amount of immobilized protein and gave some indication on the viscoelasticity of immobilized biofilms. All the modified sensors were able to specifically recognize HIV1 Vif in liquid samples. The results indicate that lower sensitivities are obtained with 4BL-V(H) single domain antibodies, possibly due to its higher hydrophobic character. The sensitivity obtained when using scFv-4BL was reestablished when using the more hydrophilic 4BL-V(H)D single domain. 4BL-V(H)D piezoimunosensors were effective in recognizing HIV1 Vif from protein mixtures and from cell extracts of human embryonic kidney cells expressing HIV1 Vif. The results presented in this paper demonstrate the potential applicability of the developed piezoimmunosensors to monitor HIV1 infection evolution.

Influence of electrolytes in the QCM response: discrimination and quantification of the interference to correct microgravimetric data.

In this work we demonstrate that the presence of electrolytes in solution generates desorption-like transients when the resonance frequency is measured. Using impedance spectroscopy analysis and Butterworth-Van Dyke (BVD) equivalent electrical circuit modeling we demonstrate that non-Kanazawa responses are obtained in the presence of electrolytes mainly due to the formation of a diffuse electric double layer (DDL) at the sensor surface, which also causes a capacitor like signal. We extend the BVD equivalent circuit by including additional parallel capacitances in order to account for such capacitor like signal. Interfering signals from electrolytes and DDL perturbations were this way discriminated. We further quantified as 8.0+/-0.5 Hz pF-1 the influence of electrolytes to the sensor resonance frequency and we used this factor to correct the data obtained by frequency counting measurements. The applicability of this approach is demonstrated by the detection of oligonucleotide sequences. After applying the corrective factor to the frequency counting data, the mass contribution to the sensor signal yields identical values when estimated by impedance analysis and frequency counting.