Bio-Photonic Sensing Cells over transparent substrates for anti-gestrinone antibodies biosensing.

In a previous work we introduced the term Bio-Photonic Sensing Cells (BICELLs), referred to periodic networks of nano-pillar suitable for biosensing when are vertically interrogated. In this article, we demonstrate the biosensing capabilities of a type of micrometric size BICELLs made of SU-8 nano-pillars fabricated over transparent substrates. We verify the biochips functionality comparing the theoretical simulations with the experimental results when are optically interrogated in transmission. We also demonstrate a sensitivity enhancement by reducing the pitch among nano-pillars from 800 to 700 nm. Thus, the Limit of Detection achievable in these types of BICELLs is in the order of 64 pg/mL for 700 nm in pitch among nano-pillars in comparison with 292 pg/mL for 800 nm in pitch when are interrogated by Fourier Transform Visible and Infrared Spectrometry. The experiments exhibited a good reproducibility with a relative standard deviation of 0.29% measured within 8 days for a specific concentration. Finally, BICELLs functionality was tested in real conditions with unpurified rabbit serum for detecting anti-gestrinone antibodies, demonstrating the high performance of this type of BICELLs to detect specific antibodies having immobilized the suitable bioreceptors onto the sensing surface.

Preparation of antibodies for the designer steroid tetrahydrogestrinone and development of an enzyme-linked immunosorbent assay for human urine analysis.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed for tetrahydrogestrinone (THG), the new designer anabolic steroid responsible for the well-known Balco scandal announced in year 2003. Antibodies have been raised against 18a-homo-pregna-4,9,11-trien-17beta-ol-3-carboxymethyl oxime coupled to horseshoe crab hemocyanin. The hapten has been synthesized from gestrinone by controlled reduction of the triple bond of the ethinyl group at position C-17, without affecting the double bonds of the steroidal rings, followed by reaction of the keto group at C-3 with (carboxymethoxy)amine hemihydrochloride to form the oxime bond. The antisera obtained has been used in combination with 18a-homo-pregna-4,9,11-trien-17beta-ol-20-yn-3-carboxymethyl oxime, a hapten derivative of gestrinone, coupled to bovine serum albumin to establish a competitive ELISA. Under the conditions used, THG can be detected in buffer with a limit of detection (LOD) of 0.045 +/- 0.015 microg L(-1) (N = 9). The assay is very selective since other steroids assessed are not recognized. Preliminary experiments performed with human urine samples demonstrate that the assay can be applied to the analysis of these samples after a simple sample treatment method reaching a LOD of 0.25 +/- 0.14 microg L(-1). Accuracy is very good as demonstrated by the excellent correlation obtained when analyzing blind spiked urine samples (slope 0.93, R2 = 0.992).