Detection of anabolic steroids in humans and animals using aptamers
Objectives:
The overall outcome of the collaborative project will be a user-friendly rapid and inexpensive tests for the reagentless semi-quantitative and quantitative detection of a battery of steroids with the only required end-user intervention being sample addition (urine / blood). The proposed project has the potential to advance significantly beyond the SoA for detection of anabolic steroids (selected from a list of testosterone and a selection of testosterone analogues) in humans and animals (both in blood and urine) using aptamers. To date no aptamers against anabolic steroids have been reported and any outcoming publications would be in high impact factor journals.
Thus the sub-objectives of the project are:
- Selection of family of aptamers for detection of anabolic steroids.
- Use of aptamers in a variety of assays including enzyme-linked oligonucleotide assay (ELONA), aptasensors and apta-RPA for ultrasensitive detection.
- Validation of selected aptamers using real human and animal samples and comparing performance with LC-MS.
Aim 1:
Selection of aptamers against testosterone and analogues of testosterone (e.g. nandrolane, stanozolol, danazol, DNA aptamers have been demonstrated to be highly specific - the best example being an aptamer that shows 10,000 times better affinity for theophylline than caffeine, despite the small molecular size and the only differentiating group being a methyl group
Aim 2:
DNA aptamers have been used in several lateral flow assays, including one for the detection of toxins, thus demonstrating the proof of concept. The selected aptamers will be used for detection of steroid metabolites in urine in a semi-quantitative lateral flow assay.
Aim 3:
Aptamers are much more flexible in their application than monoclonal antibodies and can be engineered into molecular aptamer beacons, exploiting either electrochemical of fluorescent detection. This format will allow for quantitative detection of the anabolic steroids, with the response being almost instantaneous. This format will be used for both blood (steroids) and urine (steroid metabolites) analysis. Blood analysis may need the integration of sample pre-treatment prior to detection, which can easily be achieved with a membrane.
Aim 4:
For ultrasensitive detection of extremely low levels of anabolic steroids will also be explored where aptamer detection will be combined with nucleic acid amplification in a technique developed by URV, termed apta-RPA. This test can also be incorporated within a lateral flow type assay, which can be used on-site for both blood and urine analysis. c university located in Jeddah, Kingdom of Saudi Arabia.
Aim 5:
Analytical validation of the selected aptamers and different tests developed. The current gold standard for detection of steroids is GC-LC-MS, which requires considerable technical expertise to standardise assays and interpret results. A diagnostic test for rapid and cost-effective detection of steroids in humans and animals, for use on-site is currently not available. In order to demonstrate the accuracy and reliability of the selected aptamer(s) and developed tests, a number of human and animal urine and blood samples will be analysed both by GC-LC-MS and the developed tests and the degree of correlation established.