HFF-QCM immunosensor for fast and sensitive detection of DDT in honey

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Summary

The control of chemical residues is essential in honey’s marketing to ensure consumer safety. Equipments available in the market reach the required limit of detection (LOD) (e.g. Liquid Cromatography- HPLC) but are not suitable for in-situ implantation in honey packaging industry. The reasons are:

  • High qualified staff required
  • High cost
  • Time-consuming

Other approaches available in the market, although faster and without the need of qualified staff, do not reach the required LODs. Piezoelectric immunosensors based on High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) combined with an antigen-antibody recognition process, are alternative or complementary technologies suitable for chemical residues control in food.

The analytical performance of AWS-A20 and AWS-F20 platforms (based on HFF-QCM technology) has been tested for the detection of pesticide residues (DDT) in honey.

AWS platforms lead to the following benefits:

  • Lower cost
  • Less sample/reagent consumption
  • Label-free, direct and real time transduction

DDT standard curves performed in laboratory buffer provided:

  • Sensitivity (estimated as the I50 value): 0.62 µg/L
  • LOD in the 0.05 – 0.1 µg/L range

Remarks

  • A HFF-QCM immunosensors for DDT insecticide has been developed
  • Efficient biosensing interfaces were achieved by covalent functionalization of the sensor gold electrodes with mixed alkane-thiol self-assembled monolayers (mSAMs)
  • High-sensitivity monoclonal antibodies were used as the bio-recognition element of the target analyte in a competitive immunoassay format.
  • The limits of detection (LOD) and quantification (LOQ) and the working range (WR) of the developed HFF-QCM immunosensor for DDT are in the same order of magnitude as those previously reported for ELISA and SPR immunosensor.

 

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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.

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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.