A High Fundamental Frequency (HFF)-based QCM Immunosensor for Tuberculosis Detection

Tuberculosis, one of the oldest diseases affecting human beings, is still considered as a world public health problem by the World Health Organization. Therefore, there is a need for new and more powerful analytical methods for early illness diagnosis. With this idea in mind, the development of a High Fundamental Frequency (HFF) piezoelectric immunosensor for the sensitive detection of tuberculosis was undertaken. A 38 kDa protein secreted by Mycobacterium tuberculosis was first selected as the target biomarker. Then, specific monoclonal antibodies (MAbs) were obtained. Myc-31 MAb, which showed the highest affinity to the analyte, was employed to set up a reference enzyme-linked immunosorbent assay (ELISA) with a limit of detection of 14 ng mL-1 of 38 kDa antigen. For the development of the HFF piezoelectric immunosensor, 100 MHz quartz crystals were used as transducer elements. The gold electrode surface was functionalized by covalent immobilization of the target biomarker through mixed self-assembled monolayers (mSAM) of carboxylic alkane thiols. A competitive immunoassay based on Myc-31 MAb was integrated with the transducer as sensing bio-recognition event. Reliable assay signals were obtained using low concentrations of antigen for functionalization and MAb for the competitive immunoassay. Under optimized conditions, the HFF immunosensor calibration curve for 38 kDa determination showed a limit of detection as low as 11 ng mL-1 of the biomarker. The high detectability attained by this immunosensor, in the picomolar range, makes it a promising tool for the easy, direct and sensitive detection of the tuberculosis biomarker in biological fluids such as sputum.This study was supported by COLCIENCIAS (Colombia), Project no. 13335212865) and by AWSensors (Valencia, Spain). All of the authors: A. Montoya, C. March, Y.J. Montagut, M.J. Moreno, J.J. Manclus, A. Arnau, Y. Jimenez, M. Jaramillo, P. A. Marin and R.A. Torres declare that they have no conflict of interest.Montoya Baides, Á.; March Iborra, MDC.; Montagut Ferizzola, YJ.; Moreno Tamarit, MJ.; Manclus Ciscar, JJ.; Arnau Vives, A.; Jiménez Jiménez, Y.... (2017). A High Fundamental Frequency (HFF)-based QCM Immunosensor for Tuberculosis Detection. Current Topics in Medicinal Chemistry. 17(14):1623-1630. https://doi.org/10.2174/1568026617666161104105210S16231630171

Oscillator for Biosensors based on Quartz Crystal Microbalance (QCM)

[EN] Quartz crystal is generally used in some applications as a microbalance taking advantage of its capacity to change the resonance frequency according to any surface mass change on the resonator.In this way a quartz crystal can be used as a transducer in a piezoelectric inmunosensor system in order to detect antigen-antibody bonds. An interface for Quartz Crystal Microbalances, QCM, based on an improved version of an oscillator in balanced differential configuration and its respective validation as a biosensor characterization system is introduced in this paper. The system was successfully tested in a piezoelectric inmunosensor for detecting the pesticide Carbaryl.[ES] El cristal de cuarzo generalmente es usado en aplicaciones como microbalanza, aprovechando la capacidad que presenta éste para variar su frecuencia de resonancia de acuerdo a los cambios de la densidad superficial de masa depositada en la superficie del resonador. De esta manera, un cristal de cuarzo puede ser utilizado como transductor en un sistema de inmunosensor piezoeléctrico, para detectar uniones antígeno – anticuerpo. En este artículo se presenta una interfaz para microbalanzasde cristal de cuarzo, QCM (del inglés Quartz Crystal Microbalance) basado en una versión mejorada de oscilador en configuración diferencial equilibrado y su validación como sistema de caracterización para biosensores. El sistema fue probado con éxito en un inmunosensor piezoeléctrico para la detección del plaguicida Carbaryl.Montagut Ferizzola, YJ.; García Narbón, JV.; Jiménez Jiménez, Y.; March Iborra, MDC.; Montoya Baides, Á.; Torres Villa, RA.; Arnau Vives, A. (2011). Oscilador para biosensores basado en microbalanza de cristal de cuarzo (QCM). Facultad de Ingeniería. Universidad de Antioquia. 61:160-168. http://hdl.handle.net/10251/55018S1601686

Love Wave Immunosensor for the Detection of Carbaryl Pesticide

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.The author also would like to acknowledge the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF) for their financing support through the grant of the INNPACTO 2012 project (DETECTA IPT-2012-0154-300000), J.V. Garcia's Fellowship, AP2007-03745, of FPU (Formacion de Profesorado Universitario) program, M. I. Rocha-Gaso's PhD CONACyT Fellowship and AWSensors Inc. for its collaboration.Rocha-Gaso, M.; García Narbón, JV.; García, P.; March Iborra, MDC.; Jiménez Jiménez, Y.; Francis, L.; Montoya Baides, Á.... (2014). Love Wave Immunosensor for the Detection of Carbaryl Pesticide. Sensors. 14(9):16434-16453. https://doi.org/10.3390/s140916434S1643416453149March, C., Manclús, J. J., Jiménez, Y., Arnau, A., & Montoya, A. (2009). A piezoelectric immunosensor for the determination of pesticide residues and metabolites in fruit juices. Talanta, 78(3), 827-833. doi:10.1016/j.talanta.2008.12.058Janshoff, A., Galla, H.-J., & Steinem, C. (2000). Piezoelectric Mass-Sensing Devices as Biosensors—An Alternative to Optical Biosensors? Angewandte Chemie, 39(22), 4004-4032. doi:10.1002/1521-3773(20001117)39:223.0.co;2-2Rocha-Gaso, M.-I., March-Iborra, C., Montoya-Baides, Á., & Arnau-Vives, A. (2009). Surface Generated Acoustic Wave Biosensors for the Detection of Pathogens: A Review. Sensors, 9(7), 5740-5769. doi:10.3390/s90705740Gronewold, T. M. A. (2007). Surface acoustic wave sensors in the bioanalytical field: Recent trends and challenges. Analytica Chimica Acta, 603(2), 119-128. doi:10.1016/j.aca.2007.09.056Länge, K., Rapp, B. E., & Rapp, M. (2008). Surface acoustic wave biosensors: a review. Analytical and Bioanalytical Chemistry, 391(5), 1509-1519. doi:10.1007/s00216-008-1911-5Arnau, A., Montagut, Y., García, J. V., & Jiménez, Y. (2009). A different point of view on the sensitivity of quartz crystal microbalance sensors. Measurement Science and Technology, 20(12), 124004. doi:10.1088/0957-0233/20/12/124004Montagut, Y. J., García, J. V., Jiménez, Y., March, C., Montoya, A., & Arnau, A. (2011). Frequency-shift vs phase-shift characterization of in-liquid quartz crystal microbalance applications. Review of Scientific Instruments, 82(6), 064702. doi:10.1063/1.3598340Abad, A., Primo, J., & Montoya, A. (1997). Development of an Enzyme-Linked Immunosorbent Assay to Carbaryl. 1. Antibody Production from Several Haptens and Characterization in Different Immunoassay Formats. Journal of Agricultural and Food Chemistry, 45(4), 1486-1494. doi:10.1021/jf9506904Manclús, J. J., Abad, A., Lebedev, M. Y., Mojarrad, F., Micková, B., Mercader, J. V., … Montoya, A. (2004). Development of a Monoclonal Immunoassay Selective for Chlorinated Cyclodiene Insecticides. Journal of Agricultural and Food Chemistry, 52(10), 2776-2784. doi:10.1021/jf035382hFrancis, L. A., Friedt, J.-M., Zhou, C., & Bertrand, P. (2006). In Situ Evaluation of Density, Viscosity, and Thickness of Adsorbed Soft Layers by Combined Surface Acoustic Wave and Surface Plasmon Resonance. Analytical Chemistry, 78(12), 4200-4209. doi:10.1021/ac051233hMauriz, E., Calle, A., Abad, A., Montoya, A., Hildebrandt, A., Barceló, D., & Lechuga, L. M. (2006). Determination of carbaryl in natural water samples by a surface plasmon resonance flow-through immunosensor. Biosensors and Bioelectronics, 21(11), 2129-2136. doi:10.1016/j.bios.2005.10.013Abad, A., & Montoya, A. (1997). Development of an Enzyme-Linked Immunosorbent Assay to Carbaryl. 2. Assay Optimization and Application to the Analysis of Water Samples. Journal of Agricultural and Food Chemistry, 45(4), 1495-1501. doi:10.1021/jf950691wMontagut, Y., García, J. V., Jiménez, Y., March, C., Montoya, Á., & Arnau, A. (2011). Validation of a Phase-Mass Characterization Concept and Interface for Acoustic Biosensors. Sensors, 11(5), 4702-4720. doi:10.3390/s11050470

Surface Generated Acoustic Wave Biosensors for the Detection of Pathogens: A Review

This review presents a deep insight into the Surface Generated Acoustic Wave (SGAW) technology for biosensing applications, based on more than 40 years of technological and scientific developments. In the last 20 years, SGAWs have been attracting the attention of the biochemical scientific community, due to the fact that some of these devices - Shear Horizontal Surface Acoustic Wave (SH-SAW), Surface Transverse Wave (STW), Love Wave (LW), Flexural Plate Wave (FPW), Shear Horizontal Acoustic Plate Mode (SH-APM) and Layered Guided Acoustic Plate Mode (LG-APM) - have demonstrated a high sensitivity in the detection of biorelevant molecules in liquid media. In addition, complementary efforts to improve the sensing films have been done during these years. All these developments have been made with the aim of achieving, in a future, a highly sensitive, low cost, small size, multi-channel, portable, reliable and commercially established SGAW biosensor. A setup with these features could significantly contribute to future developments in the health, food and environmental industries. The second purpose of this work is to describe the state-of-the-art of SGAW biosensors for the detection of pathogens, being this topic an issue of extremely importance for the human health. Finally, the review discuses the commercial availability, trends and future challenges of the SGAW biosensors for such applications

Piezoelectric immunosensor for the detection of 3,5,6-trichloro-2-pyridinol metabolite of the pesticide chlorpyrifos

La detección y el seguimiento analítico del insecticida organofosforado clorpirifos son necesarios a fin de que sus niveles no sobrepasen los límites permitidos por diferentes normas de organismos reguladores. En este trabajo se presenta un inmunosensor piezoeléctrico que puede determinar y cuantificar el 3,5,6-tricloro-2-piridinol (TCP), el principal metabolito del clorpirifos y del herbicida triclopir. El análisis y cuantificación del TCP se basa en un inmunoensayo competitivo con anticuerpo monoclonal. Para ello se inmovilizó, con enlace covalente, el conjugado de hapteno BSA-TS1 para TCP, sobre la superficie del electrodo de oro del cristal piezoeléctrico de cuarzo. Esta unión covalente a la superficie sensora se realizó mediante la formación de monocapas autoensambladas simples y mixtas (SAM y MSAM)). Se obtuvieron curvas patrón de calibración con cristales inmovilizados con ambos tipos, SAM y MSAM, y se compararon los resultados. Los valores de límite de detección (LOD) y el rango de trabajo obtenidos usando SAM simples fueron de 16 y 25-100 μg/L respectivamente, mientras que los valores obtenidos para estos dos parámetros empleando MSAM fueron de 9,5 y 18-74 μg/L respectivamente. En ambos casos, el cristal podía usarse alrededor de 100 ensayos sin pérdida apreciable de sensibilidad.Detection and monitoring of the organophosphate insecticide chlorpyrifos are required to comply with the limits allowed under different laws of regulatory agencies. Here we report on a piezoelectric immunosensor which can determine and quantify 3,5,6-trichloro-2-pyridinol (TCP), the main metabolite of chlorpyrifos and the herbicide triclopyr. The analysis and quantification of TCP is based on a competitive immunoassay with monoclonal antibody. Therefore, BSA-TS1hapten-conjugate for TCP was attached by a covalent bond to the gold electrode of a quartz crystal by means of both, simple and mixed self-assembled monolayers (SAM and MSAM). Standard calibration curves were obtained with both types, SAM and MSAM, and results were compared. For simple SAM the limit of detection (LOD) and working range obtained were 16 and 25-100 μg/L, respectively, whereas for MSAM were 9.5 and 18-74 μg/L, respectively. In both cases, the crystal may be used for about 100 tests without appreciable loss of sensitivity

Comparison of a monoclonal antibody-based enzyme-linked immunosorbent assay and gas chromatography for the determination of nicotine in cigarette smoke condensates

[EN] A competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies was developed to measure nicotine in smoke condensates of cigarettes. The ELISA standard curve displays a detection limit of 70 ng/mL and an effective working range of 0.3-4.0 mug of nicotine/mL. Using standard samples, ELISA results compare well with theoretical values (r = 0.997). The suitability of the method for industrial routine application was evaluated by measuring the nicotine concentration in 245 cigarette smoke condensates. When ELISA results were compared with those obtained by gas chromatography, a correlation coefficient r = 0.886 was obtained. The assay allows the specific, precise, and accurate determination of nicotine, without any significant interference from other alkaloids also present in tobacco smoke. Preliminary data suggest that the immunoassay could also be applied to other matrices, e.g., tobacco extracts. ELISA equipment requirements are minimum, simple, and low-cost, and an unskilled person could perform 150 analysis in a working day. On the basis of these features, ELISA is proposed as a promising alternative to instrumental methods for some industrial applications.Abad Fuentes, A.; Manclus Ciscar, JJ.; March Iborra, MDC.; Montoya, Á. (1993). Comparison of a monoclonal antibody-based enzyme-linked immunosorbent assay and gas chromatography for the determination of nicotine in cigarette smoke condensates. Analytical Chemistry. 65(22):3227-3231. doi:10.1021/ac00070a010S32273231652