title: Wearable platforms for women's health diagnostics
Creators: name: Clack, Kimberley A
Publisher: name: Griffith University
Year: 2024
Subjects: subject: nanotechnology
subject: women's health
subject: diagnostics
subject: wearable platforms

description: Disease has the potential to significantly impact women's overall quality of life, physical and emotional health and to even cause deaths. Cancer is recognised as one of the most devastating diseases both health-wise and economically. It is widely accepted that early cancer detection is crucial for preventing patient deaths. However, cancer detection is usually performed via painful, costly and invasive tissue-based biopsy. Liquid biopsy is a minimally invasive, more convenient and relatively painless alternative to solid tissue-based biopsy. Liquid biopsy offers circulating biomarker detection from many easily accessible bodily fluids. This thesis provides a detailed literature review of circulating exosome, CTC and ctDNA cancer biomarkers that are detectable in blood, urine or saliva. Various portable nanotechnology-based platforms are critically reviewed based on factors that assist or inhibit their overall portability. The working principle of the platforms is also discussed, as well as the future direction of the field. This literature review provides the motivation and theoretical framework for fabricating all the assays that are presented within this thesis. Two platforms for early cancer detection are presented in this thesis. Firstly, an electrochemical platform was developed for the early detection of HOTAIN lncRNA for the early detection of ovarian cancer. HOTAIR upregulation has been shown to increase the proliferation, migration, and invasiveness of ovarian cancer cells and therefore serves as a biomarker of ovarian cancer. The platform utilises capture probe-conjugated magnetic nanoparticles that specifically capture target lncRNA sequences and bring them to the surface of gold electrodes for an electrochemical readout. The electrochemical signal (in the presence of a [Fe (CN)6]3-/4- redox couple) is inversely proportional to the concentration of adsorbed lncRNA target sequences. The platform achieved a linear dynamic range of 100 nM and 1 pM for detecting pre-clinical model HOTAIR lncRNA samples (%RSD ≤ 5%, for n = 3). The platform was highly specific, showing clear distinction between HOTAIR lncRNA targets and non-specific miR-891 and miR-486 (100 nM) (%RSD ≤ 5%, for n = 3). The method was tested using ovarian cancer-specific cell lines (SKOV3 and OVCAR3) and mesothelial cell line (MeT-5A)-derived lncRNAs. Secondly, this thesis reports on a colourimetric on-bead LAMP platform for naked-eye detection of cancer-relevant DNA biomarkers. This platform uses magnetic nanoparticles to selectively capture, purify and isolate target nucleic acid sequences prior to biomolecular detection. However, this method presents an "on-bead" LAMP concept that incorporates the nanoparticles within the LAMP reaction. After the LAMP reaction, the colour of the solution remains "pink" for negative test results. For positive tests (due to successful amplification of target nucleic acids), the solution turns bright "yellow". This platform does not require heat denaturation, adsorption of nucleic acids at the gold electrode surface or electrochemical detection. This eliminates the need for a potentiostat, gold electrodes, cables and computer setup. Additionally, LAMP amplifies targets isothermally, avoiding expensive and technical thermal cycling machines. Our results demonstrate a tested detection range from 10 pM to 10 aM for spiked plasma DNA samples, (n = 3). Detection was achieved within one hour, inclusive of preparation and handling steps. The method could detect as low as 1 aM of the target and is applicable to detecting other DNA sequences after modification to the primer and capture probe sequences. However, there are inherent biological challenges that pose a barrier to early cancer diagnosis. These challenges may include biological sample complexity/heterogeneity, sample condition and low biomarker abundance, etc. In addition to the various biological challenges, physical platform challenges may include slow throughput, platform instability (such as a short shelf life), or an overall complex/ expensive set-up. As a result, there is significant room for improvement within the development of portable platforms for early cancer detection. Additionally, it may take a very long time to realise a commercial platform that is capable of early cancer detection. Noting this, this thesis provides a paradigm shift from early cancer detection, towards early pathogen detection before cancer occurs. I.e. rather than managing women's gynaecological health by detecting an incredibly difficult disease to diagnose early, research efforts can be focused towards monitoring other serious gynaecological conditions such as vaginitis. Essentially, the work of this thesis moves laterally to develop a wearable platform for vaginitis detection via Candida albicans detection. While vaginitis may not cause devastation at the same level of cancer, vaginitis is responsible for approximately 50% of all gynaecological-based healthcare visits. Vaginitis may be associated with symptoms such as dysuria, itching, thickened or malodorous discharge and general discomfort. This can cause significant stress, anxiety, embarrassment and poor quality of life. One of the greatest barriers to gynaecological health monitoring and early disease detection is the requirement for expensive laboratory equipment and trained laboratory staff. This is an especial problem within remote or developing areas that lack resources and manpower. Therefore, there is a great need for creating a portable platform for monitoring vaginal health or detecting disease. To address these facts, we developed a rapid, naked-eye-based colourimetric method for Candida albicans detection via thiolated (3' end) aptamer-conjugated gold nanoparticles. 1,3-β-D glucan molecules are a hallmark of C.albicans cell walls. Aptamers can specifically bind to target biomolecules, such as β-1,3-D-glucans, with high affinity and specificity, as well as low immunogenicity and low toxicity. Aptamers also feature relative ease of chemical synthesis, tuneable backbone modification and excellent stability over a range of ionic, pH and temperature conditions. Upon receptor-target-directed aggregation, a change in the optical properties of the aptamer-conjugated gold nanoparticles occurs - i.e., a redshift in surface plasmons or a red-to-blue naked eye detectable colour change. Our method is minimally invasive and can detect Candida albicans cells (without the need for sample purification) in a vaginal fluid simulant solution. Our method does not require blood, plasma or costly or cumbersome laboratory equipment. Our method can detect as few as 5x105 Candida albicans cells in a volume of less than 50 μL of sample solution. Our method offers an easily perceptible "blue" colour change for a positive result and a "pink" colour change for a negative result. To the best of our knowledge, no specific aptamer-based colourimetric gold nanoparticle-based platform exists for the direct detection of C.albicans in vaginal fluid. For the final work of the thesis, we present a proof-of-concept wearable platform for Candida albicans detection from samples of vaginal fluid simulant. With some modification, this platform is designed to be directly adhered to existing feminine hygiene pads or underwear. Our platform is rapid, inexpensive, user-friendly, and disposable and only requires three steps: (i) the addition of vaginal fluid onto sample pads; (ii) the addition of gold nanoparticle gel and running buffer, and (iii) naked eye detection. Our platform is underpinned by selective thiolated aptamer-based recognition of 1,3-β-D-glucan molecules. In the absence of C.albicans, wearable sample pads turn bright pink after buffer addition. In the presence of C.albicans, the wearable pads turn dark blue after buffer addition due to significant nanoparticle target-induced aggregation. We demonstrate naked-eye colourimetric detection of 4.4 x 106 C.albicans cells per mL and nanoparticle stability over a pH range of 3.0-8.0. We believe that this proof-of-concept platform has the potential to significantly impact women's health globally. It is hoped that someday, other researchers can expand on the work of this thesis to realise a commercial, wearable platform for women's health diagnostics.