High-frequency phase shift measurements greatly enhances the sensitivity of QCM immunosensors

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Authors: C. March, J.V. García, A. Sánchez, A. Arnau, Y. Jiménez, P. García, J.J. Manclús, A. Montoya.

Journal: Biosensors & Bioelectronics (2015)

In spite of being widely used for in liquid biosensing applications, sensitivity improvement of conventional (5-20MHz) quartz crystal microbalance (QCM) sensors remains an unsolved challenging task. With the help of a new electronic characterization approach based on phase change measurements at a constant fixed frequency, a highly sensitive and versatile high fundamental frequency (HFF) QCM immunosensor has successfully been developed and tested for its use in pesticide (carbaryl and thiabendazole) analysis. The analytical performance of several immunosensors was compared in competitive immunoassays taking carbaryl insecticide as the model analyte. The highest sensitivity was exhibited by the 100 MHz HFF-QCM carbaryl immunosensor. When results were compared with those reported for 9 MHz QCM, analytical parameters clearly showed an improvement of one order of magnitude for sensitivity (estimated as the I50value) and two orders of magnitude for the limit of detection (LOD): 30 μg l−1 vs 0.66 μg L−1I50 value and 11 μg L−1 vs 0.14 μg L−1 LOD, for 9 and 100 MHz, respectively. For the fungicide thiabendazole, I50 value was roughly the same as that previously reported for SPR under the same biochemical conditions, whereas LOD improved by a factor of 2. The analytical performance achieved by high frequency QCM immunosensors surpassed those of conventional QCM and SPR, closely approaching the most sensitive ELISAs. The developed 100 MHz QCM immunosensor strongly improves sensitivity in biosensing, and therefore can be considered as a very promising new analytical tool for in liquid applications where highly sensitive detection is required.

 

Further sensitivity enhancement of HFF-QCM immunosensors for pesticides

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Authors: C. March, J.V. García, R. Fernández, Y.Jiménez, A. Arnau. A.Montoya

Event: 4th International Conference on Biosensing Technology, Lisbon (2015)

Recently, High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) immunosensors have succesfully been developed. Therefore, sensitivity of QCM biosensors is no longer a drawback. Taking advantage of this previous work, we have developed a renewed highly sensitive HFF piezoelectric immunosensor using carbaryl insecticide as a model analyte for pesticide detection. To this purpose, 100 MHz quartz crystal sensors were used as the transducer elements of the biosensor and a monoclonal antibody-based competitive immunoassay was integrated as the sensing specific bio-recognition event. The biosensing interface was improved by employing mixed self-assembled monolayers (mSAMs) of alkane thiols as intermediate layers for surface functionalization. This approach allowed the covalent attachment of the assay conjugate (20.0 µg mL-¹ of BSA-CNH conjugate) onto the gold electrode surface in a more orderly and stable way than with simple SAMs. A very low concentration (1.0 µg mL-¹) of LIB-CNH45 monoclonal antibody was used for the competitive immunoassays. All immunosensor assays were performed in the AWS-A10 test platform from AWSensors. In terms of analytical performance, the new carbaryl HFF-QCM immunosensor showed higher sensitivity than the previously developed one, with analytical parameters very close to those of the most sensitive reported ELISA for carbaryl.

CLICK HERE TO DOWNLOAD THE POSTER about this work

Development of a High Fundamental Frequency (HFF) Piezoelectric Immunosensor for early and sensitive detection of tuberculosis

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Authors: A. Montoya, C. March, Y.J. Montagut, M.J. Moreno, J.J. Manclús, A. Arnau, Y. Jiménez, M. Jaramillo, P.A. Marín, R.A. Torres.

Event: International Work-Conference on Bioinformatics and Biomedical Engineering, IWBBIO 2015, Granada (Spain), April 15-17, 2015. (2015)

Tuberculosis is one of the oldest diseases affecting human beings and at present it is still considered as a world public health problem, both in developing and in developed countries. Consequently, the World Health Organization encourages the development of new and more powerful analytical methods able to sensitively detect tuberculosis biomarkers in order to provide early diagnosis and treatment.

Taking advantage of the most recent advances in new detection technologies, such as biosensors and particularly immunosensors, in the present paper we describe the development of a High Fundamental Frequency (HFF) piezoelectric immunosensor for the sensitive detection of tuberculosis. First, a secretion protein (38 kDa) from Mycobacterium tuberculosis was selected as the tuberculosis biomarker. Next, high affinity monoclonal antibodies (MAbs) specific to the target analyte were obtained. To this purpose, a recombinant form of the 38 kDa protein was used as immunizing antigen and hybridoma technology was subsequently applied for antibody production. The best MAb (namely Myc-31) was employed as the primary immunoreagent for the development of an enzyme-linked immunosorbent assay (ELISA) for 38 kDa quantification. This ELISA reached a detection limit as low as 0.014 μg mL-1 of the 38 kDa antigen and was used as a previous diagnostic test and reference assay for the future biosensor.

Finally, the HFF piezoelectric immunosensor for 38 kDa detection and quantification was developed. High fundamental frequency (100 MHz) quartz crystals were used as the transducer elements of the biosensor. Crystals were functionalized by covalent immobilization of the 38 kDa antigen onto the gold electrode surface of the quartz crystal. Mixed self-assembled monolayers (mSAM) of alkane- thiols and acids were used as intermediate layers for immobilization. A monoclonal antibody-based competitive immunoassay was then integrated as the sensing specific bio-recognition event coupled to the transducer. With this aim, the concentration of the 38 kDa antigen for immobilization and the limiting amount of the MAb for the competitive assay were previously optimized. All immunosensor assays were performed in the AWS A10 platform developed by AWSensors. As expected for HFF piezoelectric transducers, high and stable assay signals were obtained using very low protein concentrations for crystal functionalization (20 μg mL-1 of the 38 kDa antigen) and for the competitive immunoassay (0.75 μg mL-1 of Myc-31 MAb).

Under these conditions, the HFF immunosensor calibration curve for the determination of the 38 kDa antigen was performed by assaying different biomarker concentrations in the [ng mL-1 – mg mL-1] range. Each assay cycle took around 25 min including regeneration of the functionalized sensor surface between assays.

Regeneration was achieved by treatment with 0.01 M HCl. Figure 1 depicts the average 38 kDa standard curve obtained from several experiments using different 100 MHz crystals. It shows the typical decreasing sigmoidal shape associated to competitive assays.

Figure 1: Calibration curve of the HFF piezoelectric immunosensor for the 38 kDa biomarker

The IC50 value of the competitive standard curve (analyte concentration producing a 50% inhibition of the maximum assay signal, which is in turn considered as an estimate of the assay sensitivity in immunoassays) was 0.080 μg mL-1 of the 38 kDa antigen. The limit of detection of the HFF immunosensor (calculated as the analyte concentration providing a 10% inhibition of the maximum assay signal) was around 0.011 μg mL-1 of the 38 kDa biomarker, even lower than that of the previously developed ELISA. The high detectability attained by this immunosensor, in the picomolar range, makes it a very promising tool for the easy, direct and sensitive detection of the tuberculosis biomarker in biological fluids such as sputum.

Keywords: 38 kDa tuberculosis biomarker; Monoclonal antibody; ELISA; High Fundamental Frequency (HFF); Piezoelectric Immunosensor.

Publication on AWSensors technology

Love Mode Surface Acoustic Wave and High Fundamental Frequency QCM Immunosensors for the Detection of Carbaryl Pesticide

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Authors: J.V. García, M.I. Rocha, C. March, P. García, L.A. Francis, A. Montoya, A. Arnau, Y. Jiménez

Event: Eurosensors XXVIII Conference. Brescia, Italia (2014)

 

In this work we determined the Sensitivity (estimated as the I50 value) and Limit of Detection (LOD) for the immunodetection of carbaryl pesticide with two different types of acoustic wave sensors: High Fundamental Frequency QCM Quartz Crystal Microbalance (HFF-QCM) and Love Mode Surface Acoustic Wave (LM-SAW). Results were compared with others previously reported using different sensors and techniques, like traditional QCM, Surface Plasmon Resonance (SPR) and Enzyme-Linked ImmunoSorbent Assay (ELISA). We used the AWS-A10 research platform (AWSensors, Spain) to perform the experiments. We obtained I50 values of 0.31 μg/L and 0.66 μg/L, and LODs of 0.09 μg/L and 0.14 μg/L, for 120 MHz LM-SAW and 100 MHz HFF-QCM devices, respectively. Both the sensitivities and LODs of the immunosensors improved previously reported SPR and QCM results by one and two orders of magnitude, respectively, and reached those of ELISA.

Love Wave Immunosensor for the detection of carbaryl pesticide

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Authors: M. Rocha-Gaso, J.V. García, P. García, C. March, Y. Jiménez, L.A. Francis, A.I Montoya, A. Arnau

Journal: Sensors (2014)

 

A Love Wave (LW) immunosensor was developed for the detection of carbaryl pesticide. The experimental setup consisted on: a compact electronic characterization circuit based on phase and amplitude detection at constant frequency; an automated flow injection system; a thermal control unit; a custom-made flow-through cell; and Quartz /SiO2 LW sensors with a 40 μm wavelength and 120 MHz center frequency. The carbaryl detection was based on a competitive immunoassay format using LIB-CNH45 monoclonal antibody (MAb). Bovine Serum Albumin-CNH (BSA-CNH) carbaryl hapten-conjugate was covalently immobilized, via mercaptohexadecanoic acid self-assembled monolayer (SAM), onto the gold sensing area of the LW sensors. This immobilization allowed the reusability of the sensor for at least 70 assays without significant signal losses. The LW immunosensor showed a limit of detection (LOD) of 0.09 μg/L, a sensitivity of 0.31 μg/L and a linear working range of 0.14–1.63 μg/L. In comparison to other carbaryl immunosensors, the LW immunosensor achieved a high sensitivity and a low LOD. These features turn the LW immunosensor into a promising tool for applications that demand a high resolution, such as for the detection of pesticides in drinking water at European regulatory levels.

Publication on AWSensors technology

Analysis, implementation and validation of a Love mode surface acoustic wave device for its application as sensor of biological processes in liquid media

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Authors: Rocha-Gaso, M.I.; Jiménez Y. and Francis, L.

Publisher: Universitat Politècnica de València, 2013

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Comparison between High Fundamental Frequency Quartz Crystal Microbalance and Love Mode Surface Acoustic Wave devices in the detection of Carbaryl pesticide

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Authors: J.V. García, M.I. Rocha, C. March, P. García, L.A. Francis, A. Montoya, A. Arnau, Y. Jiménez

Event: Acoustic Sensors in analytical and Biophysical Studies, 29-30 August 2013, IMBB-FORTH, Heraklion, Crete.

 

In this work two acoustic technologies: High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) and Love Mode Surface Acoustic Wave (LM-SAW) have been compared for the detection of Low Molecular Weight (LMW) compounds in terms of Sensitivity and LOD. The results have also been compared with those obtained with other techniques: Traditional QCM, Surface Plasmon Resonance (SPR) and Enzyme-Linked ImmunoSorbent Assay (ELISA). Carbaryl pesticide was chosen as model analyte because it had been used as a reference LMW compound by those mentioned techniques.

AWS-A10 research platform (AWSensors, Spain) was used to perform the experiments. This platform allowed for a comparison of both devices measured by the same characterization system under similar experimental conditions.

The results achieved with LM-SAW and 100Mhz HFF-QCM were in the same order of magnitude. The achieved value sensitivity (I50 value) and LOD (I90 ) were around 0.31 µ/L and 0.09 µ/L, respectively, for LM-SAW device, and around 0’66 µ/L and 0’14 µ/L, respectively, for HFF-QCM device. Sensitivities and LODs with these novel developments on acoustic technologies improve SPR and traditional QCM technologies, and approach ELISA’s ones.

Review the poster about this experiment by clicking here:

Póster about AWS-HFF sensors for detection of Carbaryl pesticide showed at Crete 2013

 

Advanced, high sensitivity QCM piezoelectric immunosensors for pesticide analysis, based on the measurement of phase changes at high fundamental frequency (HFF)

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Authors: A. Montoya, J.J. Manclús, A. Arnau, Y. Jiménez, J.V. García-Narbón, C. March

Event:  8th MGPR International Symposium of Pesticides in Food and the Environment in Mediterranean Countries. Cappadocia,  Turkey (2013)

 

The detection of very small mass changes by means of the Quartz Crystal Microbalance (QCM) is a very popular transduction technique in biosensor design for analytical applications. In fact, QCM biosensors for pesticide analysis in fruit and derived products have recently been reported. Despite their interesting analytical performance, these biosensors still need a thorough optimization of several essential analytical parameters. In particular, sensitivity and limit of detection should be significantly improved, so that pesticide biosensors could extend their applicability to more demanding applications, such as the analysis of drinking water. In order to overcome these limitations, a new concept of piezoelectric biosensor is proposed. Unlike classic QCM, where frequency changes are detected as the response to small mass changes, the new biosensor is based on the detection of phase changes, working at high constant frequency (high fundamental frequency: HFF). On this basis, a new 100 MHz QCM immunosensor based on monoclonal antibodies for pesticide analysis has been developed. A completely new device including a dedicated PEEK chip support to attach the HFF functionalized crystals, the automatic injection system, and the electronic characterization system, has been designed and built. Carbaryl and Thiabendazole were chosen as model pesticides to assess the immunosensor performance. Specific monoclonal antibodies were used as biorecognition molecules, working in competitive immunoassays in the conjugate-coated format. Specific hapten conjugates were covalently attached to the gold surfaces of the sensor electrodes by alkanethiol self-assembled monolayers (SAM). As compared to the previous low-frequency QCM immunosensors, the sensitivity was improved by more than a one order of magnitude (I50 values in the sub-µg I-1 range), whereas the limits of detection improved by around two orders of magnitude (LODs around 0.16 µg I-1). Immunoregent consumption was decreased by 5 times (antibody) and 1000 times (assay conjugate). The high sensitivity reached by these immunosensors allows pesticide analysis at concentrations near the European MRLs for drinking water.

Un nuevo concepto de inmunosensor piezoeléctrico (QCM) para plaguicidas basado en la detección de cambios de fase a alta frecuencia

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Inmunosensor piezoeléctrico de alta sensibilidad.

Authors: A. Montoya, J.V. García-Narbón, A. Sánchez, A. Arnau, Y. Jiménez, C. March

Event: Ibersensors 2012. Puerto Rico (2012)

 

Para resolver las limitaciones de sensibilidad asociadas a los biosensores QCM basados en la detección de pequeños cambios de frecuencia, se propone un nuevo concepto de biosensor piezoeléctrico. El nuevo biosensor se basa en la detección de cambios de fase, trabajando a frecuencia alta y constante. Sobre esta base, se ha desarrollado un inmunosensor QCM de 50 MHz para la determinación de plaguicidas con alta sensibilidad.

Consulta el trabajo completo presentado en este Congreso aquí.

High-sensitivity piezoelectric immunosensor for pesticide analysis

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Authors: J.V. García, C. March, A. Sánchez, Y. Montagut, Y. Jiménez, A. Montoya, A. Arnau

Event: Biosensors 2012, 22 World Congress on Biosensors. Cancún, México (2012)

 

A 50 MHz quartz crystal microbalance (QCM) immunosensor has been developed for the determination of pesticides. High fundamental frequency (HFF) inverted mesa quartz crystal resonators were used. To provide mechanical stability and robustness quartz crystal resonators were attached permanently to a PEEK chip holder. This also allowed easy manipulation in all experimental processes. An automated flow injection analysis system was developed. The system consists of: the flow cell for the sensor chips; a flow circuit with automated syringe pumps, distribution and injection valves; and an electronic characterization system, based on the phase mass measurement at constant frequency. Carbaryl pesticide was chosen as a model analyte. Due to the low molecular weight of this insecticide, the immunoassay format chosen was a competitive one, with monoclonal antibodies (MAbs). The hapten conjugate was covalently immobilized, via mercaptohexadecanoic acid (MHA) self assembled monolayer (SAM), onto the sensor electrode surface. The immunosensor results showed an improvement of one order of magnitude in terms of sensitivity (I50 ≈ 2 μg/L) and two orders of magnitude in terms of limit of detection (LOD < 0,2 μg/L), compared with results obtained with standard 10MHz QCM sensors previously reported by our group. The automated analysis system allowed a very good reproducibility of measurements. Further improvement in terms of sensitivity and limit of detection could be easily accomplished working with higher fundamental frequency sensors (100-150 MHz), which can be directly used with the developed system.