Development of piezoelectric immunosensors based on monoclonal antibodies for pesticida analysis

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Authors: C. March, J.J. Manclús, A. Arnau, Y. Jiménez, T. Sogorb, A. Montoya

Event: IBERSENSORS 2006. Montevideo, Uruguay (2006)

 

A Monoclonal Antibody-based Piezoelectric Immunosensor for the analysis of pesticides was developed. Functionalized AT-cut 9 MHz quartz crystals integrated in a flow-through system were used as the transducer element. Pesticide detection was accomplished by competitive immunoassay in the conjugate-coated format, using specific monoclonal antibodies. Working in the microgravimetric mode, the frequency variations of the piezoelectric crystal were measured and correlated with the concentration of the pesticide in the sample. Frequency signals were inversely proportional to the pesticide concentrations, thus providing decreasing sigmoidal calibration curves. The immunosensor sensitivity (estimated as the assay I50 value) was around 30 µg/l. The limits of detection ( I90 values) were around 7 µg/l. A complete assay cycle, including regeneration, was performed in 20 min. and each sensor could be reliably reused for one hundred times. These analytical performance would allow the detection of pesticides in fruits and vegetables at European regulatory levels.

Development of monoclonal antibody-based piezoelectric immunosensor for pesticide analysis

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Authors: C. March, J.J. Manclús, A. Arnau, Y. Jiménez, T. Sogorb, A. Montoya

Event: 7th Workshop on Biosensors and Bioanalytical μ-Techniques in Environmental and Clinical Analysis. Kusadasi, Turkey, 10-14 September (2006)

 

Immunosensors are novel analytical devices in which the extreme selectivity provided by immunological interactions is combined with the high sensitivity achieved by electronic signal transducers. In the present work, the development of a Monoclonal Antibody-based Piezoelectric Immunosensor for the analysis of pesticides (carbaryl and the chlorpyrifos and triclopyr metabolite TCP) is described, and its main analytical characteristics are presented.

Specific carbaryl and TCP haptens were conjugated to a carrier protein (BSA) and then covalently attached to gold-coated AT-cut 9 MHz  quartz crystals, previously functionalized with a self-assembled alkanethiol (thioctic acid) monolayer. The activated piezoelectric crystal was inserted in a homemade Arnite cell that allowed only one face of the crystal to be in contact with the reagents. The cell was integrated in a flow-through system and pesticide detection was subsequently accomplished by competitive immunoassay in the conjugate-coated format. The assay consisted of a previous incubation of the analyte sample with its specific monoclonal antibody, followed by the immunoreaction of the analyte-antibody mixture with the hapten derivative immobilized on the sensor surface. The frequency variations of the piezoelectric crystal working in the microgravimetric mode were measured with a commercial Research Quartz Crystal Microbalance (RQCM) and correlated with the concentration of the respective pesticide in the sample. The total time required for a complete assay cycle, including regeneration, was 20 min.

As it was expected for binding inhibition immunoassays, the frequency signals provided by the sensor were inversely proportional to the pesticide concentrations. Sigmoidal calibration curves were obtained by fitting experimental points to a four-parameter logistic equation. The immunosensor sensitivity, expressed as the analyte concentration that reduced the assay signal by 50% (I50) around 25 µg/l for carbaryl and 32 µg/l for TCP. The limits of detection, calculated as the pesticide concentrations that provide 90% of the maximum signal, were around 6 and 9 µg/l, respectively. This analytical performance would allow the detection of carbaryl and TCP in fruits and vegetables at European regulatory levels. For both pesticides, the hapten-functionalized piezoelectric crystals could be reused for more than one-hundred times with only slight reductions of the maximum signal and without significant losses of sensitivity.

 

Analog-Digital Phase-Locked Loop for Alternating Current Quartz Electrogravimetry

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Authors: R. Torres, A. Arnau, H. Perrot, J. García, C. Gabrielli

Journal: Electronics Letters, Vol 42, N. 22, pp 1272-1273 (2006)

 

Requirements for frequency–voltage conversion in alternating current (AC) electrogravimetry are introduced. A new frequency–voltage conversion system based on a double tuning analogue–digital phaselocked loop is proposed. The reported results prove its reliability for AC electrogravimetry measurements.

Introduction: AC electrogravimetry is based on an electrochemical quartz crystal microbalance (EQCM) used in dynamic regime. In EQCM one of the deposited gold electrodes of the quartz crystal resonator can be coated with an electroactive polymer film and can be used as the working electrode (WE) following a classical electrochemical configuration. The frequency shift of the quartz crystal microbalance (QCM) allows obtaining of the mass response associated with the charge transfer, which occurs at polymer=electrolyte interface. AC electrogravimetry was proposed to characterise and separately identify ions and solvent motion at the film=electrolyte interface. In this technique the mass response to a small potential perturbation is analysed in the frequency domain thanks to a fast QCM used in dynamic regime; for that, a continuous voltage with a superimposed small potential sinusoidal perturbation is applied between the reference electrode and the WE of the electrochemical cell. Thus, the so-called electrogravimetric transfer function (EGTF), defined as the ratio (Dm=DE) between the amplitude of induced mass change (Dm) and the perturbation amplitude (DE), can be plotted in a complex plane for the entire range of perturbation frequencies. The various species involved are characterised by a loop in the complex plane and can be separately identified when the loops do not overlap.

A contribution to solve the problem of coating properties extraction in Quartz Crystal Microbalance Applications

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Authors: Y. Jiménez, R. Fernández, R. Torres, A. Arnau

Journal: IEEE Transactions on ultrasonics, Ferroelectrics and Frequency control. Vol.53, Iss 5, pp 1057-1072 (2006)

The problem of coating properties extraction in quartz crystal resonator (QCR) applications is one of the challenging tasks of QCR applications, not completely solved even in theoretical conditions. The present work demonstrates that the problem is determined only for a set of three parameters of the coating, which concentrate the four coating properties. The following parameters are chosen: the surface mass density, the loss tangent and the magnitude of the characteristic impedance. An algorithm is proposed that permits, in ideal conditions, an unambiguous extraction of these parameters, starting only from the admittance spectrum of a three-layer compound resonator, obtained from the one-dimensional transmission-line model (TLM) around the resonance. The algorithm introduces three additional improvements in relation to other routines: the calculation time is drastically reduced, the problem of erroneous solutions related to relative minima in typical fitting routines is eliminated, and a systematic error analysis in the extraction of the coating properties in real practice can be carried out. The use of the proposed algorithm as a tool for studying the effects of different phenomena such as slipping or surface roughness is introduced.