Carmen March

A piezoelectric immunosensor for the determination of pesticide residues and metabolites in fruit juices

A quartz crystal microbalance (QCM) immunosensor was developed for the determination of the insecticide carbaryl and 3,5,6-trichloro-2-pyridinol (TCP), the main metabolite of the insecticide chlorpyrifos and of the herbicide triclopyr. The detection was based on a competitive conjugate-immobilized immunoassay format using monoclonal antibodies (MAbs). Hapten conjugates were covalently immobilized, via thioctic acid self-assembled monolayer (SAM), onto the gold electrode sensitive surface of the quartz crystal. This covalent immobilization allowed the reusability of the modified electrode surface for at least one hundred and fifty assays without significant loss of sensitivity. The piezoimmunosensor showed detection limits (analyte concentrations producing 10% inhibition of the maximum signal) of 11 and 7 microg l(-1) for carbaryl and TCP, respectively. The sensitivity attained (I(50) value) was around 30 microg l(-1) for both compounds. Linear working ranges were 15-53 microg l(-1) for carbaryl and 13-83 microg l(-1) for TCP. Each complete assay cycle took 20 min. The good sensitivity, specificity, and reusability achieved, together with the short response time, allowed the application of this immunosensor to the determination of carbaryl and TCP in fruits and vegetables at European regulatory levels, with high precision and accuracy.

Validation of a Phase-Mass Characterization Concept and Interface for Acoustic Biosensors

Acoustic wave resonator techniques are widely used in in-liquid biochemical applications. The main challenges remaining are the improvement of sensitivity and limit of detection, as well as multianalysis capabilities and reliability. The sensitivity improvement issue has been addressed by increasing the sensor frequency, using different techniques such as high fundamental frequency quartz crystal microbalances (QCMs), surface generated acoustic waves (SGAWs) and film bulk acoustic resonators (FBARs). However, this sensitivity improvement has not been completely matched in terms of limit of detection. The decrease on frequency stability due to the increase of the phase noise, particularly in oscillators, has made it impossible to increase the resolution. A new concept of sensor characterization at constant frequency has been recently proposed based on the phase/mass sensitivity equation: Δφ/Δm ≈ −1/mL, where mL is the liquid mass perturbed by the resonator. The validation of the new concept is presented in this article. An immunosensor application for the detection of a low molecular weight pollutant, the insecticide carbaryl, has been chosen as a validation model.

Frequency-shift vs phase-shift characterization of in-liquid quartz crystal microbalance applications.

The improvement of sensitivity in quartz crystal microbalance (QCM) applications has been addressed in the last decades by increasing the sensor fundamental frequency, following the increment of the frequency/mass sensitivity with the square of frequency predicted by Sauerbrey. However, this sensitivity improvement has not been completely transferred in terms of resolution. The decrease of frequency stability due to the increase of the phase noise, particularly in oscillators, made impossible to reach the expected resolution. A new concept of sensor characterization at constant frequency has been recently proposed. The validation of the new concept is presented in this work. An immunosensor application for the detection of a low molecular weight contaminant, the insecticide carbaryl, has been chosen for the validation. An, in principle, improved version of a balanced-bridge oscillator is validated for its use in liquids, and applied for the frequency shift characterization of the QCM immunosensor application. The classical frequency shift characterization is compared with the new phase-shift characterization concept and system proposed.

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

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 100MHz HFF-QCM carbaryl immunosensor. When results were compared with those reported for 9MHz QCM, analytical parameters clearly showed an improvement of one order of magnitude for sensitivity (estimated as the I50 value) and two orders of magnitude for the limit of detection (LOD): 30μgl(-1) vs 0.66μgL(-1)I50 value and 11μgL(-1) vs 0.14μgL(-1) LOD, for 9 and 100MHz, 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 100MHz 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.

Electrochemical assays based on enzyme-electrode systems to determine glycerol and propylene glycol in tobacco casing

Abstract Two enzymatic electrochemical methods for the determination of glycerol and propylene glycol in tobacco casing are described. Amperometric determination of glycerol is accomplished by the consecutive action of two coupled enzymes (glycerokinase and glycerol-3-phosphate oxidase) which metabolize the analyte and consume oxygen proportionally to glycerol concentration. Oxygen consumption is measured with a Clark-type electrode. Potentiometric determination of propylene glycol is based on its enzymatic degradation by the enzyme glycerol dehydrogenase in a reaction in which H + ions are produced. The subtle increase in H + concentration (pH decrease), which is proportional to propylene glycol concentration, is measured by a pH electrode. The signals generated by the electrodes are picked up and transferred to a personal computer by a high performance communication/data conversion card. Using original software, the system interpolates data from samples in a surface of standard curves performed by plotting oxygen concentration or pH decrease versus glycerol or propylene glycol concentration, respectively. The working range is 60–300 μM and precise and accurate determinations are carried out in 2 to 3 min. Good correlations are found between the results obtained with the enzymatic sensor prototypes and those obtained by a reference analytical technique (gas chromatography).

A new QCM sensor characterization technique based on the phase/mass sensitivity concept

Quartz crystal microbalance (QCM) technique is widely used in in-liquid biochemical applications. The main challenges remain on the improvement of sensitivity and limit of detection, as well as multianalysis capabilities and reliability. The improvement of sensitivity has been addressed in the last decades by increasing the sensor fundamental frequency; following the increment of the frequency/mass sensitivity with the square of frequency predicted by Sauerbrey. However, this sensitivity improvement has not been completely transferred in terms of limit of detection. A new concept of sensor characterization has been recently proposed based on the phase/mass sensitivity equation: Δφ/Δm ≈−1/mL, where mL is the liquid mass perturbed by the resonator in its oscillatory movement; this mass reduces proportionally with the frequency square root. The validation of the new concept is presented in this work. An immunosensor application for the detection of the very low molecular weight insecticide, carbaryl, has been chosen for the validation of the characterization technique.

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

BACKGROUND Tuberculosis, one of the oldest diseases affecting human beings, is still considered as a world public health problem by the World Health Organization. METHOD & MATERIAL 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. RESULTS & DISCUSSION 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.