Detection of DDT and carbaryl pesticides in honey by means of immunosensors based on high fundamental frequency quartz crystal microbalance (HFF-QCM).

BACKGROUND: In recent years there has been a concern about the presence of pesticides in honey because residues of DDT and carbaryl were found in honey samples. Traditional techniques, such as chromatography, reach the required limits of detection (LOD) but are not suitable for in situ implementation in the honey-packaging industry due to their high cost and the need for highly qualified staff for routine operation. Biosensors offer simplicity, low cost, and easy handling for analytical purposes in food applications. RESULTS: Piezoelectric immunosensors based on high fundamental frequency quartz crystal microbalance (HFF-QCM) have been developed for the detection of carbaryl and DDT in honey. Biorecognition was based on competitive immunoassays in the conjugate-coated format, using monoclonal antibodies as specific immunoreagents. The assay LODs attained by the HFF-QCM immunosensors were 0.05 µg L-1 for carbaryl and 0.24 µg L-1 for DDT, reaching a similar level of detectability to that of the usual reference techniques. The practical LODs in honey samples were 8 µg kg-1 for carbaryl and 24 µg kg-1 for DDT. The immunosensors' analytical performance allow the detection of these pesticides in honey at EU regulatory levels with good accuracy (recovery percentages ranging from 94% to 130% within the working range of each pesticide standard curve) and precision (coefficients of variation in the 9-36% range). CONCLUSION: The proposed immunosensor is a promising analytical tool that could be implemented for quality control in the honey packaging industry, to simplify and to reduce the cost of the routine pesticide analysis in this appreciated natural food. © 2020 Society of Chemical Industry.

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.

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.

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/m(L), where m(L) 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.

Rapid detection and counting of viable beer-spoilage lactic acid bacteria using a monoclonal chemiluminescence enzyme immunoassay and a CCD camera.

A chemiluminescence enzyme immunoassay carried out with a monoclonal antibody (MAb) and a charge-coupled device (CCD) camera was developed for rapid enumeration of viable beer-spoilage lactic acid bacteria. LA-4 MAb, which recognizes a broad spectrum of lactic acid bacteria isolated from several breweries across Spain, was produced and characterized. Test samples were filtered through polycarbonate membranes, and the membranes with retained bacteria were incubated at 31 degrees C for 2 days. They were then subjected to a two-step chemiluminescence enzyme immunoassay with MAb LA-4, and light-emitting points were detected and counted with a CCD camera. Eighteen out of 19 beer-spoilage lactic acid bacteria analysed produced luminous spots that could be enumerated. Results provided by the immunochemiluminescence assay correlated very well with those obtained by visual plate counting within a range of 3-100 CFU/100 ml. Correlation coefficients were 0.994 for four strains in sterile saline solution and 0.984 for 14 strains in artificially contaminated beer. The excellent agreement suggests that luminous spots detected within 2 days of culture are produced only by viable cells.