Rapid detection of anabolic steroids in urine by protein arrays

The purpose of this study was to develop a rapid and sensitive method utilizing the state-of-the-art protein arrays technique to detect urinary anabolic steroids (ASs) in athletes. Three experiments were designed to investigate the feasibility of the protein arrays for ASs testing. Firstly, androgen receptor (AR) and estrogen receptor (ER) protein arrays were prepared on polysaccharide-coated slides to investigate whether they can bind to ASs (affinity tests). Secondly, in comparison to adrenergic receptor (the receptor of beta-blockers) and opioid receptor (the receptor of narcotic analgesics) arrays, AR and ER protein arrays were used to test whether they can determine the ASs positive urine sample specifically (specific binding tests). At last protein arrays were used to estimate qualitatively the ASs in positive urine samples (qualitative tests). From the results of the affinity tests the shape of the dose-dependence curve suggested a positive cooperative binding of ASs with the protein arrays. The AR and ER protein arrays showed affinities for fluorescence labelled testosterone and estradiol that were similar to those of literatures (0.65 vs. 0.89 nM, 5.96 vs. 10.3 nM, respectively). Based on the data, the sensitivity of testing can reach 0.1 nM that was much better than the World Anti-Doping Code (WADA) standard. Specific binding tests showed that the prohibited substance in positive urine samples belonged to the anabolic estrogenic inhibitor of ASs. From the results of qualitative tests, we could estimate that there were anabolic androgenic steroids in the positive urine samples and their concentration was lower than 50 microM Methyltestosterone. The total time of the test process for ASs in urine needed less than 1 h. In summary, the present study showed that the protein arrays method provided a highly sensitive and rapid alternative to screen urine samples for the detection of the misuse of ASs in athletes and was suitable for testing in both weekly training sessions as well as large-scale competition events.