A fluorimetric assay for cortisol

Abstract

A simple, rapid and sensitive fluorimetric assay for the quantitative determination of cortisol is reported. The assay is based on the formation of a fluorescent dye when cortisol is incubated with a mixture of sulfuric acid and acetic acid. The fluorescence spectrum recorded for the resulting dye shows a maximum extinction at 475 nm and a maximum emission at 525 nm. The solvent 2-methyl-4-pentanone was used for extraction and was found to act as a fluorescence amplifier. A limit of detection of 2.7 muM was achieved, making it possible to forego solvent evaporation. The assay suffers minor interference from 11-deoxycortisol which exhibits low fluorescence at lambda (ex): 460 nm; lambda (em): 505 nm. Typical standard deviations were below 4%. We validated the assay using a biotransformation with recombinant Schizosaccharomyces pombe which regioselectively hydroxylates 11-deoxycortisol to cortisol. The method described herein is suitable for preliminary screening of microorganisms capable of steroid hydroxylation.

Molecular Evolution of a Steroid Hydroxylating Cytochrome P450 Using a Versatile Steroid Detection System for Screening

“…For example, monoclonal cortisol antibodies show a cross reactivity of 19% with RSS [23]. We used a fluorescence assay based on a method described by Appel et al [10]. Steroids exhibit fluorescence in acidic environment in which the intensity of the fluorescence depends on the numbers of hydroxyl groups [25].…”

“…We decided to use a fluorescence assay due to its high sensitivity and ease of handling. For this the previously described activity assay [10,11] was adopted to micro titer plates and high-throughput conditions. Tests were performed to assess the general usability of the fluorescence assay for our purposes (Fig.…”

“…In contrast, conventional detection methods for steroids, like HPLC and GC-MS, are time consuming and quite expensive. Appel et al [10] presented a simple detection method for steroids. They used a fluorescence based test system to determine the conversion rate of 11-deoxycortisol to cortisol of a CYP11B1 expressing Schizosaccharomyces pombe culture.…”

Prostate Cancer in African-American Men: Serum Biomarkers for Early Detection Using Nanoparticles

“…Upon protein binding, changes in the electrical properties in the vicinity of an electrode are detected. Electrochemical sensors is a new and promising group of sensing devices, allowing increased sensitivities in conjunction with lower cost, low analy sis tim es, miniaturized and affo rdable platform s [17][18][19][20][21][22][23][24]. There are nu merous detection techniques, currently being research ed towards an a pplication in electroche mical sensors, which are de scribed in detail elsewhere [25][26][27][28][29][30][31][32][33][34].…”

Rapid micromotor-based naked-eye immunoassay

“…Cortisol, an important stress and clinical biomarker [15][16][17][18][19], was selected as model target to demonstrate the capabilities of this new approach. The most widely used methods in clinical assays of cortisol include luminescence and fluorescence assays [20,21], enzyme-linked immunosorbent assays (ELISAs) [22], and immunoassays in connection to fluorescence [23] or electrochemical detection [17,[24][25][26][27][28]. However, these cortisol-detection methods still present some limitations in terms of analysis time, pre-treatment steps, and cost and do not involve rapid naked-eye detection.…”