Development of piezoelectric immunosensors based on monoclonal antibodies for pesticida analysis

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

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

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

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.

 

A monoclonal antibody-based piezoelectric immunosensor for the analysis of carbaryl

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

Event: 4th MGPR International Symposium of pesticides in food and the environment in Mediterranean Countries. Pine Bay Holiday Resort Kusadasi-Aydin. Turkey (2005)

 

Immunosensors combine immunoassay selectivity with the high sensitivity often provided by electronic signal transducers. In this communication, the development of a monoclonal antibody-based Piezoelectric Immunosensor for the analysis of carbaryl is described and coupled to BSA were covalently attached to gold coated AT- cut 9 MHz quartz crystals, previously functionalized with a self-assembled alkanethiol monolayer. The 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 accomplished by competitive immunoassay in the conjugate-coated format. The assay consisted of the incubation of the analyte with a specific monoclonal antibody, followed by the immunoreaction of the mixture with the carbaryl derivative immobilized on the sensor surface. The frequency variations of the piezoelectric crystal were measured with a commercial Research Quartz Crystal Microbalance (RQCM) and correlated with the carbaryl concentration in the standards. The total time required for a complete assay cycle, including regeneration, was 20 min. As it corresponds to binding inhibition immunoassays, the frequency signal provided by the sensor was inversely proportional to the analyte concentration. A sigmoidal calibration curve was obtained by fitting experimental points to a four-parameter equation. The immunoassay sensitivity, expressed as the carbaryl concentration that reduced the assay signal by 50% (I50), was around 25 µg/l. The inhibition curve would allow the detection of carbaryl in fruits and vegetables at European regulatory levels.

Viscoelastic characterization of electrochemically prepared conducting polymer films by impedance analysis at Quartz Crystal. Study of the surface roughness effect on the effective values of the thickness and viscoelastic properties of the coating

Authors: Y. Jiménez, R.Torres, A. Arnau, M.Otero, E. Forzani, E.J. Calvo

Event: XVIII International Symposium on Biolectrochemistry and Bioenergetics of the Bioelectrochemical society in conjuction with 3rd spring meeting of the International Society of Electrochemistry (ISE). Coimbra, Portugal (2005)

 

An Electrochemical Quartz Crystal Microbalance is used for a continuous monitoring of the growth of the polymer poly(3,4-ethylenedioxy) thiopene tetrabutyammonium perchlorate (PEDT-TBAP), which is electro-polymerized in acetonitrile on a gold electrode of a 10 MHz quartz crystal resonator. The surface acoustic impedance of the resonator is analyzed starting from the electrical admittance continuously measured by means of a network analyzer. Changes in the acoustic impedance suggest that a mechanical resonance phenomenon occurs during the electro-deposition of the coating. In order to determine the origin of this effect, the evolution of the physical properties of the coating is analyzed. The extraction of the physical properties of the coating is carried out by means of an algorithm recently developed by the authors. Four sets of coating properties are obtained with the algorithm in four different conditions. In one of them an additional technique, ellipsometry, is used to have an alternative measurement of the coating thickness; no additional techniques are necessary in the rest of the cases. All the cases show a significant change in the viscoelastic properties of the coating during the time-interval of the suspected mechanical resonance. Changes in the surface roughness of the coating during the experiment are confirmed by scanning electron microscopy (SEM). The analysis of the surface roughness of the coating and its effect on the effective values of the coating properties, seem to indicate that the changes in the viscoelastic properties are due to the changes in the surface roughness of the coating, and that these changes are responsible for the mechanical resonance effect. This analysis is carried out by means of the same algorithm which is used, in this case, as a simulation tool.

Viscoelastic characterization of electrochemically prepared conducting polymer films by impedance analysis at Quartz Crystal. Study of the surface roughness effect on the effective values of the thickness and viscoelastic properties of the coating

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

Event: 5th edition of Acoustic Wave Based Sensors: Fundamentals, Concepts, New Applications. Physikzentrum Bad Honnef, Germany (2005)

 

An Electrochemical Quartz Crystal Microbalance is used for a continuous monitoring of the growth of the polymer poly(3,4-ethylenedioxy) thiopene tetrabutyammonium perchlorate (PEDT-TBAP), which is electro-polymerized in acetonitrile of a gold electrode of a 10 MHz quartz crystal resonator. The surface acoustic impedance of the resonator is analyzed starting from the electrical admittance continuously measured by means of a network analyzer. Changes in the acoustic impedance suggest that a mechanical resonance phenomenon occurs during the electro-deposition of the coating. In order to determine the origin of this effect, the evolution of the physical properties of the coating is analyzed. The extraction of the physical properties of the coating is carried out by means of an algorithm recently developed by the authors. Four sets of coating properties are obtained with the algorithm in four different conditions. In one of them an additional technique, ellipsometry, is used to have an alternative measurement of the coating thickness, no additional techniques are necessary in the rest of the cases. All the cases show a significant change in the viscoelastic properties of the coating during the time-interval of the suspected mechanical resonance. Changes in the surface roughness of the coating during the experiment are confirmed by scanning electron microscopy (SEM). The analysis of the surface roughness of the coating and its effect on the effective values of the coating properties, seem to indicate that the changes in the viscoelastic properties are due to the changes in the surface roughness of the coating, and that these changes are responsible for the mechanical resonance effect. This analysis is carried out by means of the same algorithm which is used, in this case, as a simulation tool.

 

 

A new roughness physical model for describing the mechanical impedance of a coated shear resonator immersed in liquids

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

Event: 5th edition of Acoustic Wave Based Sensors: Fundamentals, Concepts, New Applications. Physikzentrum Bad Honnef, Germany (2005)

 

The models that describe the surface acoustic impedance at the surface of a coated AT quartz sensor should include the effects of the roughness, both the roughness of the crystal surface and the roughness of the coating. When these effects  are not kept in mind, the values of the physical magnitudes extracted starting from the measurable characteristic parameters of the sensor, usually the complete admittance spectrum around resonance or the frequency and motional resistance shifts, can be erroneous. In this work a model that facilitates the understanding of the physical phenomena that happen when a rough surface oscillates in contact with a fluid is presented. This model characterizes the roughness like a surface of spheroids of variable magnitudes. The solution of the Navier-Stokes equation applied to this allows the obtaining of the acoustic impedance of the rough surface in contact with the liquid, and even to extend the pattern to those cases in which the rough layer has viscoelastic properties.

Systematic error analysis in the determination of physical parameters of the coating in Quartz Crystal Resonator sensors

Authors: Y. Jiménez, A. Arnau, T. Sogorb, M. Otero, E. Calvo

Event: 4th Acoustic Wave Sensor Workshop. Salbris, France (2003)

 

A new algorithmic strategy which permits an unambiguous extraction of the effective surface mass density, loss tangent and characteristic impedance of the first coating layer in AT cut quartz crystal resonators (QCR) is introduced. A preliminary analysis of the propagating error in the determination of these effective physical parameters, due to practical errors in the admittance measuring system or due to a lack of precision in the admittance model for describing the real admittance of the sensor, is presented as well. This analysis makes clear the necessity of a standard indicating the specifications of the measuring system including the cell interface for an appropriate sensor characterization.

Circuit for continuous motional series resonant frequency and motional resistance monitoring of quartz crystal resonators by parallel capacitance compensation

Authors: A. Arnau, T. Sogorb, Y. Jiménez

Event: Acusticum, Sevilla, Spain (2002)

 

A deep analysis of the problem associated to interface circuits for quartz-crystal-microbalance sensors reveals that the so-called static capacitance of the sensor is one of the elements which make the use of oscillators more critical for sensor applications. A phase-locked-loop based circuit designed for compensating the parallel capacitance in quartz crystal resonator sensors is presented. This circuit permits the calibration of the external circuitry to the sensor and provides a continuous measurement of the motional series resonant frequency and motional resistance. An extension and automation of the proposed system for multiple sensor characterization is introduced. Experimental results are also shown.