Label-free immunosensor for monitoring vitellogenin as a biomarker for exogenous oestrogen compounds in amphibian species

Abstract

A label-free, optical waveguide lightmode spectroscopy based immunosensor was developed for frog (Bombina orientalis) vitellogenin (Vtg) determination in biological samples as a biomarker for exogenous oestrogen compounds. Antibody against Vtg was produced in rabbits immunised with purified lipovitellin (Lpv), a precursor of Vtg, from the homogenised ovary of oriental fire-bellied toads (B. orientalis). The purified protein and Lpv/Vtg-specific serum were applied in both competitive and direct immunoassay formats using optical waveguide lightmode spectroscopy immunosensor. When measuring Vtg in direct manner, the Lpv antibody (1.76 µg mL −1 ) was immobilised on the sensor surface, and the linear measuring range for Vtg was 0.1-10 µg mL −1 . During the competitive measurement, 100 ng mL −1 Lpv was applied for the immobilisation. The linear measuring range for Vtg was 0.5-50 ng mL −1 . We studied the relative substrate specificity of the antibody, and it was concluded that the method is suitable for the sensitive and selective determination of Vtg levels in toads. Heart, liver and gonad samples from male animals were spiked with Vtg and were analysed using the newly developed method, and female toads and spawn samples were tested and compared to the calibration curve obtained by the spiked samples.

Direct and Competitive Optical Grating Immunosensors for Determination of Fusarium Mycotoxin Zearalenone

“…Detectable analyte concentrations in assay buffer were found to be five orders of magnitude lower by the immunosensor than by the related ELISA method, which, considering the sample preparation requirements, corresponds to a three orders of magnitude improvement for determination of ZON content in maize commodity. Such unique improvements in the analytical sensitivity of the OWLS technique compared to the corresponding ELISA method have previously been evidenced for the detection of other analytes, including a nearly three orders of magnitude enhancement for the endocrine biomarker protein vitellogenin [ 77 ] and a six orders of magnitude improvement for a herbicide active ingredient trifluralin [ 76 ]. Moreover, the current OWLS immunosensor represents substantial advancements compared to previous immunosensors for ZON, e.g., based on SPR [ 71 ], TIRE [ 73 ], and electrochemical detection with antibodies immobilised on gold nanoparticles embedded on multi-walled carbon nanotubes [ 42 ], to which the LOD of the current competitive OWLS immunosensor represents 30,000-, 5000-, and 75-fold improvements, respectively.…”

“…Immunosensors based on the technique of optical waveguide lightmode spectroscopy (OWLS) have been applied with success to detect different molecules, and gained importance in environmental and food analysis [ 59 , 76 , 77 ]. In the current study, an OWLS immunosensor has been developed for the determination of ZON in maize samples.…”

Nanophotonic label-free biosensors for environmental monitoring

“…The biosensors show similar or improved sensitivities than standard ELISA test, and specially, the OWLS biosensor for ochratoxin A detection reaches a better LOD compared with the MZI biosensor previously described [14]. The OWLS biosensors have also been applied for the detection of vitellogenin, an egg yolk protein precursor used as biomarker of endocrine disrupting effects in carp [21,22] and amphibian species [23].…”

OWLS Based Nanosensors for Agro-Environmental and Food Safety

“…The competitive immunosensor allowed a sensitive detection range for Lpv between 3 and 100 ng ml -1 . To monitor the EDC pollution of the specific environmental regions, an OWLS based immunosensor was also developed for the determination of Vtg from the Oriental fire-bellied toad (Bombina orientalis) [11].…”