An aptamer-based sensing platform for luteinising hormone pulsatility measurement

Abstract

Normal fertility in human involves highly orchestrated communication across the hypothalamic-pituitary-gonadal (HPG) axis. The pulsatile release of Luteinising Hormone (LH) is a critical element for downstream regulation of sex steroid hormone synthesis and the production of mature eggs. Changes in LH pulsatile pattern have been linked to hypothalamic dysfunction, resulting in multiple reproductive and growth disorders including Polycystic Ovary Syndrome (PCOS), Hypothalamic Amenorrhea (HA), and delayed/precocious puberty. Therefore, assessing the pulsatility of LH is important not only for academic investigation of infertility, but also for clinical decisions and monitoring of treatment. However, there is currently no clinically available tool for measuring human LH pulsatility. The immunoassay system is expensive and requires large volumes of patient blood, limiting its application for LH pulsatility monitoring.

In this thesis, I propose a novel method using aptamer-enabled sensing technology to develop a device platform to measure LH pulsatility. I first generated a novel aptamer binding molecule against LH by a nitrocellulose membrane-based in vitro selection then characterised its high affinity and specific binding properties by multiple biophysical/chemical methods. I then developed a sensitive electrochemical-based detection method using this aptamer. The principal mechanism is that structure switching upon binding is associated with the electron transfer rate changes of the MB redox label. I then customised this assay to numerous device platforms under our rapid prototyping strategy including 96 well automated platform, continuous sensing platform and chip-based multiple electrode platform. The best-performing device was found to be the AELECAP (Automated ELEctroChemical Aptamer Platform) – a 96-well plate based automatic micro-wire sensing platform capable of measuring a series of low volume luteinising hormone within a short time.

Clinical samples were evaluated using AELECAP. A series of clinical samples were measured including LH pulsatility profile of menopause female (high LH amplitude), normal female/male (normal LH amplitude) and female with hypothalamic amenorrhea (no LH pulsatility). Total patient numbers were 12 of each type, with 50 blood samples collected every 10 mins in 8 hours. Results showed that the system can distinguish LH pulsatile pattern among the cohorts and pulsatility profiles were consistent with the result measured by clinical assays.

AELECAP shows high potential as a novel approach for clinical aptamer-based sensing. AELECAP competes with current automated immunometric assays system with lower costs, lower reagent use, and a simpler setup. There is potential for this approach to be further developed as a tool for infertility research and to assist clinicians in personalised treatment with hormonal therapy.