Fast and Accurate Determination of Minute Ochratoxin A Levels in Cereal Flours and Wine with the Label-Free White Light Reflectance Spectroscopy Biosensing Platform.

Ochratoxin A (OTA) is one of the most toxic naturally encountered contaminants and is found in a variety of foods and beverages, including cereals and wine. Driven by the strict regulations regarding the maximum allowable OTA concentration in foodstuff and the necessity for on-site determination, the development of fast and sensitive methods for the OTA determination in cereal flours and wine samples, based on white light reflectance spectroscopy, is presented. The method relied on appropriately engineered silicon chips, on top of which an OTA-protein conjugate was immobilized. A polyclonal antibody against OTA was then employed to detect the analyte in the framework of a competitive immunoassay; followed by the subsequent addition of a biotinylated secondary antibody and streptavidin for signal enhancement. A small size instrument performed all assay steps automatically and the bioreactions were monitored in real time as the software converted the spectral shifts into effective biomolecular adlayer thickness increase. The assay developed had a detection limit of 0.03 ng/mL and a working range up to 200 ng/mL. The assay lasted 25 min (less than 1h, including calibrators/antibody pre-incubation) and was accomplished following a simple sample preparation protocol. The method was applied to corn and wheat flour samples and white and red wines with recovery values ranging from 87.2 to 111%. The simplicity of the overall assay protocol and convenient instrumentation demonstrates the potential of the immunosensor developed for OTA detection at the point of need.

Fast, sensitive and selective determination of herbicide glyphosate in water samples with a White Light Reflectance Spectroscopy immunosensor.

An optical immunosensor based on White Light Reflectance Spectroscopy is described for the determination of the herbicide glyphosate in drinking water samples. The biosensor allows for the label-free real-time monitoring of biomolecular interactions taking place onto a SiO2/Si chip by transforming the shift in the reflected interference spectrum caused by the immunoreaction to effective biomolecular adlayer thickness. Glyphosate determination is accomplished by functionalizing the chip with a protein conjugate of the herbicide followed by a competitive immunoassay format. Prior to the assay, glyphosate derivatization in the calibrators and/or the samples was performed through reaction with succinic anhydride. Under the optimized assay protocol, a detection limit of 10 pg mL-1 was achieved. Recovery values ranging from 90.0 to 110% were determined in spiked bottled and tap water samples, demonstrating the accuracy of the method. In addition, the sensor could be regenerated and re-used for at least 14 times without statistically significant effect on the assay sensitivity and accuracy. The excellent analytical performance and short analysis time (approx. 25 min), combined with the small sensor size, should be helpful for the fast on-site determination of glyphosate in drinking water samples.

Multiplexed mycotoxins determination employing white light reflectance spectroscopy and silicon chips with silicon oxide areas of different thickness.

Biosensing through White Light Reflectance Spectroscopy (WLRS) is based on monitoring the shift of interference spectrum due to the binding reactions occurring on top of a thin SiO2 layer deposited on a silicon chip. Multi-analyte determinations were possible through scanning of a single sensor chip on which multiple bioreactive areas have been created. Nonetheless, the implementation of moving parts increased the instrumentation size and complexity and limited the potential for on-site determinations. Thus, in this work, a new approach, which is based on patterning the sensor surface to create areas with different SiO2 thickness, is developed and evaluated for multi-analyte determinations with the WLRS set-up. The areas of different thickness can be interrogated by a single reflection probe placed on a fixed position over the chip and the reflection spectrum recorded is de-convoluted to the spectra corresponding to each area allowing the simultaneous monitoring of the bioreactions taking place at each one of them. The combination of different areas thickness was optimized using chips with two areas for single analyte assays. The optimum chips were then used for the simultaneous determination of two mycotoxins, aflatoxin B1 and fumonisin B1. A competitive immunoassay format was followed employing immobilization of mycotoxin-protein conjugates onto the SiO2 of different thickness. It was found that the dual-analyte assays had identical analytical characteristics with the respective single-analyte ones. The detection limits achieved were 0.05 ng/mL for aflatoxin B1 and 1.0 ng/mL for fumonisin B1, with dynamic ranges extending up to 5.0 and 50 ng/mL, respectively. The sensor was also evaluated for the determination of the two mycotoxins in whole grain samples (wheat and maize). The extraction protocol was optimized and recoveries ranging from 85 to 115% have been determined. Due to lack of moving parts, the novel multi-analyte format is expected to considerably facilitate the built-up of a portable device for determination of analytes at the point-of-need.

Detection of DNA mutations using a capacitive micro-membrane array.

The detection of DNA hybridization using capacitive readout and a biosensor array of ultrathin Si membranes is presented. The biosensor exploits the ability of the ultrathin membranes to deflect upon surface stress variations caused by biological interactions. Probe DNA molecules are immobilized on the membrane surface and the surface stress variations during hybridization with their complementary strands force the membrane to deflect and effectively change the capacitance between the flexible membrane and the fixed substrate. The sensor array comprises 256 such sensing sites thus allowing the concurrent sensing of multiple DNA mutations. The biosensor and its performance for the detection of complementary DNA strands are demonstrated using beta-thalassemia oligonucleotides. The experimental results show that the presented sensors are able to detect DNA hybridization and to discriminate single nucleotide mismatches.