Biomimetic estrogen sensor based on soft colloidal probes.

An increasing number of reports substantiate the link between emerging estrogenic pollutants and a variety of adverse effects including developmental disorders, infertility, cancer and neurological disorders, threatening public health as well as environment. The detection of the diverse classes of estrogenic and antiestrogenic substances is still challenging due to analytics which needs to cover the whole range of compounds acting on estrogen receptors and the complex estrogen pathways. In this proof-of-concept study, we report a novel biomimetic detection scheme based on the specific recognition of estrogenic ligands by estrogen sulfotransferase 1E1 (SULT1E1), which acts as one of the key enzymes in estrogen homeostasis. SULT1E1 was site-specifically immobilized on transparent glass slides via a hexahistidine-tag in a multi-step procedure. Soft colloidal probes (SCPs) covalently functionalized with ligands of SULT1E1, namely estrone and estradiol 17-(ß-D-glucuronide), served as adhesion probes. The various functionalization steps were analyzed and optimized using epifluorescence, confocal laser scanning as well as reflection interference contrast microscopy (RICM). A competitive SCP binding assay probing the elastic SCP deformation driven by the specific interaction between SCPs and the SULT1E1 decorated glass slides was employed in conjunction with an optical readout by RICM and automated image analysis to detect estrogenic compounds by their inhibition of SCP adhesion. This sensing concept has demonstrated exceptional specificity for estrogenic steroid compounds compared to structurally related substance classes and provides promising options for multiplexed assays and incorporation of other proteins of the endocrine system to fully capture the whole ensemble of hormonally active substances.

Picomolar glyphosate sensitivity of an optical particle-based sensor utilizing biomimetic interaction principles.

The continually growing use of glyphosate and its critically discussed health and biodiversity risks ask for fast, low cost, on-site sensing technologies for food and water. To address this problem, we designed a highly sensitive sensor built on the remarkably specific recognition of glyphosate by its physiological target enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs). This principle is implemented in an interferometric sensor by using the recently established soft colloidal probe (SCP) technique. EPSPs was site-specifically immobilized on a transparent surface utilizing the self-assembling properties of circadian clock gene 2 hydrophobin chimera and homogeneity of the layer was evidenced by atomic force microscopy. Exposure of the enzyme decorated biochip to glyphosate containing samples causes formation of enzyme-analyte complexes and a competitive loss of available binding sites for glyphosate-functionalized poly(ethylene glycol) SCPs. Functionalization of the SCPs with different types of linker molecules and glyphosate was assessed employing confocal laser scanning microscopy as well as confocal Raman microspectroscopy. Overall, reflection interference contrast microscopy analysis of SCP-biochip interactions revealed a strong influence of linker length and glyphosate coupling position on the sensitivity of the sensor. In employing a combination of pentaglycine linker and tethering glyphosate via its secondary amino group, concentrations in aqueous solutions down to 100 pM could be measured by the differential adhesion between SCP and biochip surface, supported by automated image analysis algorithms. This sensing concept could even prove its exceptional pM sensitivity in combination with a superior discrimination against structurally related compounds.

Surface Functionalization by Hydrophobin-EPSPS Fusion Protein Allows for the Fast and Simple Detection of Glyphosate.

Glyphosate, the most widely used pesticide worldwide, is under debate due to its potentially cancerogenic effects and harmful influence on biodiversity and environment. Therefore, the detection of glyphosate in water, food or environmental probes is of high interest. Currently detection of glyphosate usually requires specialized, costly instruments, is labor intensive and time consuming. Here we present a fast and simple method to detect glyphosate in the nanomolar range based on the surface immobilization of glyphosate's target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) via fusion to the hydrophobin Ccg2 and determination of enzyme activity with a malachite green assay, which is a common photometric technique to measure inorganic phosphate (Pi). The assay demonstrates a new approach for a fast and simple detection of pesticides.